CN115915956A - Feed composition - Google Patents

Abstract

Engineered robust high Tm phytase clade polypeptides and fragments thereof are described herein. Methods of making such engineered robust high Tm phytase clades and fragments thereof are also described, as well as uses of such engineered robust high Tm phytase clades and fragments thereof for enhancing animal performance.

Description

Feed composition

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/982,944, filed on February 28, 2020, the disclosure of which is incorporated herein by reference in its entirety.

Incorporation by Reference

The sequence listing provided in the form of a file of 324KB in size and named "20210224_NB41804-WO-PCT_Sequence Listing_ST25", created and concurrently filed on February 24, 2021, is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to feed compositions and premixes containing engineered robust high Tm phytase clade polypeptides and fragments thereof, and methods of producing such feed compositions and premixes to enhance animal performance.

Background Art

Phytase is the most commonly used exogenous enzyme in monogastric animal feeds. Phytase can reduce the anti-nutritional effects of phytate and improve the digestibility of phosphorus, calcium, amino acids and energy, as well as reduce the negative impact of inorganic phosphorus excretion on the environment.

Phytate is the major storage form of phosphorus in cereals and legumes. However, monogastric animals such as pigs, poultry and fish cannot effectively metabolize or absorb phytate (or phytic acid) in their diets and therefore excrete it, leading to phosphorus pollution in areas of intensive livestock production. In addition, phytic acid also acts as an anti-nutrient in monogastric animals by chelating metal agents such as calcium, copper and zinc, and forming insoluble complexes with proteins and amino acids in various sections of the digestive tract.

It has long been believed that non-ruminant animals lack endogenous phytases and are therefore unable to utilize phytate. However, endogenous mucosal phosphatases and bacterial phytases have been described as active in the small intestine and cecum of poultry. Maenz, D.D.; Classen, H.L., Phytase activity in the small intestinal brush border membrane of the chicken. Poult Sci 1998, 77, 557-63. Abudabos, A.M., Phytate phosphorus utilization and intestinal phytase activity in laying hens. Italian Journal of Animal Science 2012, 11, e8. Zeller, E.; Schollenberger, M.; Kuhn, I.; Rodehutscord, M. In order to provide these animals with adequate phosphate for growth and health, inorganic phosphates are added to their diets. Such additions can be expensive and further increase pollution problems.

Phytates are usually hydrolyzed by the action of phytases to produce lower inositol phosphates and inorganic phosphates. Phytases are used as additives to animal feeds, where they improve the availability of organic phosphorus to the animals and reduce phosphate pollution of the environment (Wodzinski R J, Ullah A H. Adv Appl Microbiol. [Progress in Applied Microbiology] 42, 263-302 (1996)).

Fungi (Wyss M. et al., Appl. Environ. Microbiol. 65 (2), 367-373 (1999); Berka R. M. et al., Appl. Environ. Microbiol. 64 (11), 4423-4427 (1998); Lassen S. et al., Appl. Environ. Microbiol. 67 (10), 4701-4707 (2001)) and bacteria (Greiner R. et al., Arch. Biochem. Biophys. 303 (1), 107-113 (1993); Kerovuo et al., Appl. Environ. Microbiol. 64 (6), 2079-2085 (1998); Kim et al., Appl. Environ. Microbiol. 64 (6), 2079-2085 (1998); Kim et al., Appl. Environ. Microbiol. 67 (10), 4701-4707 (2001)) have been described in the literature. H.W. et al., Biotechnol. Lett. 25, 1231-1234 (2003); Greiner R. et al., Arch. Biochem. Biophys. 341(2), 201-206 (1997); Yoon S.J. et al., Enzyme and microbial technol. 18, 449-454 (1996); Zinin N.V. et al., FEMS Microbiol. Lett. 236, 283-290 (2004)).

US Patent No. 8,053,221 issued on November 8, 2011 to Miasnikov et al. relates to phytases derived from the bacterium Buttiauxella species, and variants/modified forms thereof selected and/or engineered for improved characteristics compared to the wild-type (parent) enzyme.

US Patent No. 6,110,719 issued to Short on August 29, 2000 and US Patent No. 6,183,740 issued to Short et al. on February 6, 2001 relate to phytases derived from Escherichia coli B.

U.S. Patent No. 9,365,840 issued on June 4, 2016 to Sjoeholm et al. relates to polypeptides having phytase activity.

U.S. Patent No. 8,206,962, issued to Lassen et al. on June 26, 2011, and U.S. Patent No. 8,507,240, issued to Lassen et al. on August 13, 2013, are directed to Hafnia phytase variants.

U.S. Patent No. 8,557,552 issued on October 15, 2013 to Haefner et al. relates to synthetic phytase variants.

WO 2015/012890, with an International Publication Date of January 29, 2015, relates to polypeptides having phytase activity.

A new generation of phytases has been developed over the past decade. However, these phytases, when applied to feeds in liquid form before conditioning and granulation, do not have the appropriate robustness to withstand high levels of stress under commercially relevant feed granulation conditions. Therefore, the heat-stable phytase products suitable for commercial granulation on the market are dry products, and many have protective coatings to maintain activity. However, it is desirable to apply phytases in liquid form to feeds, because, for example, phytases added in liquid form will be evenly distributed and immediately released in animals when delivered by feed. There is still a need for such phytases and fragments thereof, which are robust when applied in liquid form before conditioning and granulation under commercially relevant conditions and can still improve animal performance.

Summary of the invention

In some aspects, provided herein is an animal feed pellet or premix comprising:

An engineered phytase polypeptide or a fragment thereof comprising phytase activity, wherein the phytase polypeptide or the fragment thereof has at least 82% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and

Liquid or solid carrier. In certain embodiments, the carrier comprises one or more of water, glycerol, glyceride, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol. In certain embodiments, the carrier is one or more hydrocolloids selected from the group consisting of alginate, gelatin, cellulose derivatives, polysaccharides, molasses and brewer's grains (vinasses). In certain embodiments, the carrier is one or more of the following: molasses, protamolasses, brewer's grains, liquid fermentation byproducts, liquid corn steep liquor, liquid wheat wine lees, liquid corn wine lees, liquid barley wine lees, grain wine lees, liquid corn gluten powder, liquid byproducts from ethanol processing, liquid byproducts from grain processing, liquid byproducts from gluten production. In certain embodiments, the carrier can be melted. In certain embodiments, the carrier is one or more carriers selected from the group consisting of: animal oil or fat, vegetable oil or fat, triglyceride, free fatty acid, animal wax, beeswax, lanolin, shell wax, insect wax, vegetable wax, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic wax, natural and synthetic resins and mixtures thereof. In certain embodiments, the fatty acid is one or more selected from the group consisting of: medium chain fatty acid (MCFA), lauric acid, C8+C10 mixture, butyric acid, lactic acid, propionic acid, formic acid and succinic acid. In certain embodiments, the fat is animal fat or oil and/or vegetable fat or oil. In certain embodiments, the vegetable fat or oil is selected from the group consisting of: canola oil, cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil, tung oil, castor oil and rapeseed oil. In some embodiments, the vegetable fat or oil is selected from the group consisting of: fully hardened palm oil, fully hardened rapeseed oil, fully hardened cottonseed oil and fully hardened soybean oil. In some embodiments of any embodiment disclosed herein, the vegetable fat or oil is palm oil or fully hardened palm oil. In some embodiments, the liquid carrier is one or more of liquid whey, liquid lactose-free whey, liquid acid whey, liquid milk, liquid milk from industrial cleaning, liquid processed milk. In some embodiments of any embodiment disclosed herein, the carrier is one or more of lecithin, lecithin glycerol mixture or lecithin fatty acid mixture. In some embodiments, the carrier is one or more compounds selected from the group consisting of: lysine, lysine sulfate, methionine, threonine, valine, tryptophan, arginine, histidine, isoleucine, leucine and phenylalanine. In some embodiments, the carrier is methionine. In some embodiments, the methionine is in the form of L-methionine, or in the form of a synthetic methionine source, such as OLM (i.e., DL-methionine) or all its salt forms, its analogs (e.g., 2-hydroxy-4-methylthiobutyric acid or all its salt forms), its derivatives (e.g., 2-hydroxy-4-methylthiobutyric acid isopropyl ester or any other ester), or mixtures thereof. In some embodiments of any of the embodiments disclosed herein, the carrier is a hydrolysate of protein. In some embodiments of any of the embodiments disclosed herein, the carrier is a liquid carrier. In some embodiments of any of the embodiments disclosed herein, the carrier is a solid carrier. In some embodiments of any of the embodiments disclosed herein, the pellets further comprise vitamins and/or minerals. In some embodiments of any of the embodiments disclosed herein, the engineered phytase polypeptide or fragment thereof is in granular form.

In other aspects, the present invention provides a method for producing animal feed pellets or premixes, the method comprising combining (a) an engineered phytase polypeptide or a fragment thereof comprising phytase activity, the phytase polypeptide or fragment thereof having at least 82% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and (b) a liquid or solid carrier. In some embodiments, the carrier comprises one or more of water, glycerol, glycerides, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol. In some embodiments, the carrier is one or more hydrocolloids selected from the group consisting of alginate, gelatin, cellulose derivatives, polysaccharides, molasses and brewer's grains. In some embodiments, the carrier is one or more of the following: molasses, raw molasses, brewer's grains, liquid fermentation byproducts, liquid corn steep liquor, liquid wheat grains, liquid corn grains, liquid barley grains, grain grains, liquid corn gluten meal, liquid byproducts from ethanol processing, liquid byproducts from grain processing, and liquid byproducts from gluten production. In some embodiments, the carrier can be melted. In certain embodiments, the carrier is one or more carriers selected from the group consisting of: animal oil or fat, vegetable oil or fat, triglyceride, free fatty acid, animal wax, beeswax, lanolin, shell wax, insect wax, vegetable wax, carnauba wax, candelilla wax, bayberry wax, sugarcane wax, mineral wax, synthetic wax, natural and synthetic resins and mixtures thereof. In certain embodiments, the fatty acid is one or more selected from the group consisting of: medium chain fatty acid (MCFA), lauric acid, C8+C10 mixture, butyric acid, lactic acid, propionic acid, formic acid and succinic acid. In certain embodiments, the fat is animal fat or oil and/or vegetable fat or oil. In certain embodiments, the vegetable fat or oil is selected from the group consisting of: canola oil, cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil, tung oil, castor oil and rapeseed oil. In some embodiments, the vegetable fat or oil is selected from the group consisting of: fully hardened palm oil, fully hardened rapeseed oil, fully hardened cottonseed oil and fully hardened soybean oil. In some embodiments of any embodiment disclosed herein, the vegetable fat or oil is palm oil or fully hardened palm oil. In some embodiments, the liquid carrier is one or more of liquid whey, liquid lactose-free whey, liquid acid whey, liquid milk, liquid milk from industrial cleaning, liquid processed milk. In some embodiments of any embodiment disclosed herein, the carrier is one or more of lecithin, lecithin glycerol mixture or lecithin fatty acid mixture. In some embodiments, the carrier is one or more compounds selected from the group consisting of: lysine, lysine sulfate, methionine, threonine, valine, tryptophan, arginine, histidine, isoleucine, leucine and phenylalanine. In some embodiments, the carrier is methionine. In some embodiments, the methionine is in the form of L-methionine, or in the form of a synthetic methionine source, such as OLM (i.e., DL-methionine) or all its salt forms, its analogs (e.g., 2-hydroxy-4-methylthiobutyric acid or all its salt forms), its derivatives (e.g., 2-hydroxy-4-methylthiobutyric acid isopropyl ester or any other ester), or mixtures thereof. In some embodiments of any of the embodiments disclosed herein, the carrier is a hydrolysate of protein. In some embodiments of any of the embodiments disclosed herein, the carrier is a liquid carrier. In some embodiments of any of the embodiments disclosed herein, the carrier is a solid carrier. In some embodiments of any of the embodiments disclosed herein, the method further comprises combining vitamins and/or minerals. In some embodiments of any of the embodiments disclosed herein, the engineered phytase polypeptide or fragment thereof is in granular form. In some embodiments of any of the embodiments disclosed herein, the method further comprises (c) granulating the combination of phytase and carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-1BB (column A to 1BB) shows the HMM probability score for each position of the polypeptide sequence of the high Tm phytase clade. The comprehensive score (COMP) of the HMM is shown in bold in the top 3 columns of Figure 1A. The position (P) and consensus sequence (consensus) (C) of each amino acid are shown in the 1st column under P/C.

FIG. 2 depicts a phylogenetic tree showing the relatedness among various phytases, including the engineered phytase polypeptides and fragments thereof described herein, based on amino acid sequence similarities and differences.

Figure 3 depicts the three-dimensional structures of representative high Tm clade phytases, modeled using the published crystal structure of the closely related Hafnia alvei 6-phytase and displayed as a ribbon diagram.

The following sequence complies with 37 C.F.R. §§1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and conforms to World Intellectual Property Organization (WIPO) Standard ST.25 (2009), and Rules 5.2 and 49.5(a-bis) of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT), and Section 208 of the Administrative Regulations and Annex C regarding sequence listings. The symbols and formats used for nucleotide and amino acid sequence data follow the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 corresponds to the predicted mature sequence of the engineered phytase PHY-13594.

SEQ ID NO: 2 corresponds to the predicted mature sequence of the engineered phytase PHY-10931.

SEQ ID NO: 3 corresponds to the predicted mature sequence of the engineered phytase PHY-10957.

SEQ ID NO: 4 corresponds to the predicted mature sequence of the engineered phytase PHY-11569.

SEQ ID NO: 5 corresponds to the predicted mature sequence of the engineered phytase PHY-11658.

SEQ ID NO: 6 corresponds to the predicted mature sequence of the engineered phytase PHY-11673.

SEQ ID NO: 7 corresponds to the predicted mature sequence of the engineered phytase PHY-11680.

SEQ ID NO: 8 corresponds to the predicted mature sequence of the engineered phytase PHY-11895.

SEQ ID NO: 9 corresponds to the predicted mature sequence of the engineered phytase PHY-11932.

SEQ ID NO: 10 corresponds to the predicted mature sequence of the engineered phytase PHY-12058.

SEQ ID NO: 11 corresponds to the predicted mature sequence of the engineered phytase PHY-12663.

SEQ ID NO: 12 corresponds to the predicted mature sequence of the engineered phytase PHY-12784.

SEQ ID NO: 13 corresponds to the predicted mature sequence of the engineered phytase PHY-13177.

SEQ ID NO: 14 corresponds to the predicted mature sequence of engineered phytase PHY-13371

SEQ ID NO: 15 corresponds to the predicted mature sequence of the engineered phytase PHY-13460.

SEQ ID NO: 16 corresponds to the predicted mature sequence of the engineered phytase PHY-13513.

SEQ ID NO: 17 corresponds to the predicted mature sequence of the engineered phytase PHY-13637.

SEQ ID NO: 18 corresponds to the predicted mature sequence of the engineered phytase PHY-13705.

SEQ ID NO: 19 corresponds to the predicted mature sequence of the engineered phytase PHY-13713.

SEQ ID NO: 20 corresponds to the predicted mature sequence of the engineered phytase PHY-13747.

SEQ ID NO: 21 corresponds to the predicted mature sequence of the engineered phytase PHY-13779.

SEQ ID NO: 22 corresponds to the predicted mature sequence of the engineered phytase PHY-13789.

SEQ ID NO: 23 corresponds to the predicted mature sequence of the engineered phytase PHY-13798.

SEQ ID NO: 24 corresponds to the predicted mature sequence of the engineered phytase PHY-13868.

SEQ ID NO: 25 corresponds to the predicted mature sequence of the engineered phytase PHY-13883.

SEQ ID NO: 26 corresponds to the predicted mature sequence of the engineered phytase PHY-13885.

SEQ ID NO: 27 corresponds to the predicted mature sequence of the engineered phytase PHY-13936.

SEQ ID NO: 28 corresponds to the predicted mature sequence of the engineered phytase PHY-14004.

SEQ ID NO: 29 corresponds to the predicted mature sequence of the engineered phytase PHY-14215.

SEQ ID NO: 30 corresponds to the predicted mature sequence of the engineered phytase PHY-14256.

SEQ ID NO: 31 corresponds to the predicted mature sequence of the engineered phytase PHY-14277.

SEQ ID NO: 32 corresponds to the predicted mature sequence of the engineered phytase PHY-14473.

SEQ ID NO: 33 corresponds to the predicted mature sequence of the engineered phytase PHY-14614.

SEQ ID NO: 34 corresponds to the predicted mature sequence of the engineered phytase PHY-14804.

SEQ ID NO: 35 corresponds to the predicted mature sequence of the engineered phytase PHY-14945.

SEQ ID NO: 36 corresponds to the predicted mature sequence of the engineered phytase PHY-15459.

SEQ ID NO: 37 corresponds to the predicted mature sequence of the engineered phytase PHY-16513.

SEQ ID NO:38 corresponds to Buttiauxella noackiae WP064555343.1.

SEQ ID NO: 39 corresponds to Citrobacter braakii AAS45884.1.

SEQ ID NO: 40 corresponds to the bacterium RDH40465.1 of the family Coxiellaceae.

SEQ ID NO: 41 corresponds to Enterobacteriaceae WP094337278.1.

SEQ ID NO:42 corresponds to E. coli WP 001297112.

SEQ ID NO:43 corresponds to Hafnia alvei WP 072307456.1.

SEQ ID NO:44 corresponds to Rouxiella badensis WP 084912871.1.

SEQ ID NO: 45 corresponds to Serratia sp. WP 009636981.1.

SEQ ID NO:46 corresponds to Yersinia aldovae WP 004701026.1.

SEQ ID NO:47 corresponds to Yersinia frederiksenii WP050140790.1.

SEQ ID NO:48 corresponds to Yersinia kristensenii WP004392102.1.

SEQ ID NO: 49 corresponds to Yersinia mollaretii WP 049646723.1.

SEQ ID NO: 50 corresponds to Yersinia rohdei WP 050539947.1.

SEQ ID NO:51 corresponds to SEQ ID NO:3 in EP 322271.

SEQ ID NO:52 corresponds to SEQ ID NO:2 in US 8101391.

SEQ ID NO:53 corresponds to SEQ ID NO:4 in US 8101391.

SEQ ID NO:54 corresponds to SEQ ID NO:35 in US 8101391.

SEQ ID NO:55 corresponds to SEQ ID NO:49 in US 8101391.

SEQ ID NO:56 corresponds to SEQ ID NO:1 in US 8143046.

SEQ ID NO:57 corresponds to SEQ ID NO:3 in US 8143046.

SEQ ID NO:58 corresponds to SEQ ID NO:2 in US 8460656.

SEQ ID NO:59 corresponds to SEQ ID NO:13 in US 8557555.

SEQ ID NO:60 corresponds to SEQ ID NO:24 in US 8557555.

SEQ ID NO:61 corresponds to SEQ ID NO:3 in US 20160083700.

SEQ ID NO:62 corresponds to SEQ ID NO:1 in WO 2010034835-0002.

SEQ ID NO:63 corresponds to the T. reesei aspartic protease signal sequence.

SEQ ID NO: 64 corresponds to the predicted mature sequence of the engineered phytase PHY-16812.

SEQ ID NO: 65 corresponds to the predicted mature sequence of the engineered phytase PHY-17403.

SEQ ID NO: 66 corresponds to the predicted mature sequence of the engineered phytase PHY-17336.

SEQ ID NO: 67 corresponds to the predicted mature sequence of the engineered phytase PHY-17225.

SEQ ID NO: 68 corresponds to the predicted mature sequence of the engineered phytase PHY-17186.

SEQ ID NO: 69 corresponds to the predicted mature sequence of the engineered phytase PHY-17195.

SEQ ID NO: 70 corresponds to the predicted mature sequence of the engineered phytase PHY-17124.

SEQ ID NO: 71 corresponds to the predicted mature sequence of the engineered phytase PHY-17189.

SEQ ID NO: 72 corresponds to the predicted mature sequence of the engineered phytase PHY-17218.

SEQ ID NO: 73 corresponds to the predicted mature sequence of the engineered phytase PHY-17219.

SEQ ID NO: 74 corresponds to the predicted mature sequence of the engineered phytase PHY-17204.

SEQ ID NO: 75 corresponds to the predicted mature sequence of the engineered phytase PHY-17215.

SEQ ID NO: 76 corresponds to the predicted mature sequence of the engineered phytase PHY-17201.

SEQ ID NO: 77 corresponds to the predicted mature sequence of the engineered phytase PHY-17205.

SEQ ID NO: 78 corresponds to the predicted mature sequence of the engineered phytase PHY-17224.

SEQ ID NO: 79 corresponds to the predicted mature sequence of the engineered phytase PHY-17200.

SEQ ID NO: 80 corresponds to the predicted mature sequence of engineered phytase PHY-17198.

SEQ ID NO: 81 corresponds to the predicted mature sequence of the engineered phytase PHY-17199.

SEQ ID NO: 82 corresponds to the predicted mature sequence of the engineered phytase PHY-17214.

SEQ ID NO: 83 corresponds to the predicted mature sequence of engineered phytase PHY-17197.

SEQ ID NO: 84 corresponds to the predicted mature sequence of the engineered phytase PHY-17228.

SEQ ID NO: 85 corresponds to the predicted mature sequence of the engineered phytase PHY-17229.

SEQ ID NO: 86 corresponds to the predicted mature sequence of the engineered phytase PHY-17152.

SEQ ID NO: 87 corresponds to the predicted mature sequence of the engineered phytase PHY-17206.

SEQ ID NO:88 corresponds to the Buttiauxella NCIMB 41248 N-terminus.

SEQ ID NO:89 corresponds to the N-terminus of Citrobacter braakii AAS45884.

SEQ ID NO:90 corresponds to the N-terminus of E. tarda YP007628727.

SEQ ID NO:91 corresponds to the N-terminus of PHY-13594.

SEQ ID NO:92 corresponds to the N-terminus of PHY-13789.

SEQ ID NO:93 corresponds to the N-terminus of PHY-13885.

SEQ ID NO:94 corresponds to the C-terminal SEQ ID NO:1 in WO 2010034835-0002.

SEQ ID NO:95 corresponds to the C-terminus of Yersinia morganii WP032813045.

SEQ ID NO:96 corresponds to Buttiauxella NCIMB 41248 C-terminus.

SEQ ID NO:97 corresponds to the C-terminus of PHY-13594.

SEQ ID NO:98 corresponds to the C-terminus of PHY-13789.

SEQ ID NO:99 corresponds to the C-terminus of PHY-13885.

SEQ ID NO: 100 corresponds to the PHY-13594 core region.

SEQ ID NO: 101 corresponds to the PHY-13789 core region.

SEQ ID NO: 102 corresponds to the PHY-13885 core region.

SEQ ID NO: 103 corresponds to the PHY-16812 core region.

SEQ ID NO:104 corresponds to SEQ ID NO:4 in US 7081563.

DETAILED DESCRIPTION

All patents, patent applications, and publications cited are incorporated herein by reference in their entirety.

In this disclosure, a number of terms and abbreviations are used. Unless otherwise specifically stated, the following definitions apply.

As used herein, the singular forms "a/an" and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. For example, the terms "a/an", "the", "one or more", and "at least one" may be used interchangeably herein.

The terms "and/or" and "or" are used interchangeably herein and refer to a specific disclosure that each of two specified features or components has or does not have the other. Thus, the term "and/or" as used in the phrase "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and/or" as used in the phrase "A, B, and/or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Words using the singular include the plural and vice versa.

The terms "comprises/comprising", "includes/including", "having" and variations thereof are used interchangeably and mean "including but not limited to". It should be understood that wherever an aspect is described with the language "comprising", similar aspects described with the terms "consisting of" and/or "consisting essentially of" are also provided.

The term "consisting of" means "including and limited to."

The term "consisting essentially of" means the specified materials of a composition or the specified steps of a process, as well as those additional materials or steps that do not materially affect the basic characteristics of the materials or process.

Throughout the application, various embodiments can be presented in the form of range.It should be understood that the description of range form is only for convenience and simplicity, and should not be construed as an inflexible limitation of the scope of embodiments described herein.Therefore, the description of scope should be considered to have all possible sub-ranges disclosed and the individual numerical values in the scope.For example, the description of scope such as 1 to 6 should be considered to have the individual numerical values disclosed in the scope, such as 1 to 2, 1 to 3, 1 to 4 and 1 to 5, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6 etc., and the individual numerical values in the scope, for example 1, 2, 3, 4, 5 and 6.No matter how the width of the scope, this is applicable.

As used herein, the term "about" can allow for a certain degree of variability in values or ranges, for example, within 10%, within 5%, or within 1% of a stated value or stated limit of a range.

The term "phytase" (inositol hexaphosphate phosphohydrolase) refers to a class of phosphatases that catalyze the hydrolysis of phytic acid (inositol hexaphosphate or IP6), an indigestible organic form of phosphorus found in cereals and oil seeds, and release inorganic phosphorus in a usable form.

The terms "animal" and "subject" are used interchangeably herein and refer to any organism belonging to the kingdom Animalia, and include, but are not limited to, mammals (excluding humans), non-human animals, domestic animals, livestock, farm animals, zoo animals, breeding stock, and the like. For example, all non-ruminants and ruminants may be mentioned. In one embodiment, the animal is a non-ruminant, i.e., a monogastric animal. Examples of monogastric animals include, but are not limited to, pigs (pigs and swine), such as piglets, growing pigs, sows; poultry, such as turkeys, ducks, chickens, broilers, laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans, such as shrimp and prawns. In further embodiments, the animal is a ruminant, including, but not limited to, cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns, and wildebeests.

The term "clade", also called a monophyletic group, refers to a group of organisms or related sequences that share a common ancestor and all of its direct descendants.

The term " _Tm " is the temperature at which a protein denatures, or the free energies of the unfolded and folded states are equal and half the population is unfolded and the other half is folded. The thermal unfolding behavior of enzymes is often studied using calorimetry or optical techniques such as circular dichroism, fluorescence or light scattering.

The term "high Tm phytase clade" refers to a clade of phytase polypeptides or fragments thereof having a Tm of at least 92.5° using differential scanning calorimetry as described below in Example 3. The terms "high Tm phytase clade polypeptides" and "engineered phytase polypeptides and fragments thereof" are used interchangeably herein.

The terms "mixer liquid application" and "MLA" are used interchangeably herein and refer to animal feed production in which heat-sensitive compounds, particularly enzymes, can be applied to animal feed in liquid form prior to conditioning and pelleting and remain functional in the feed after conditioning and pelleting.

"Feed" means any natural or artificial diet, meal, etc., or components of such a meal, which is intended or suitable for consumption, ingestion, digestion, respectively, by a non-human animal. Preferably, the term "feed" is used to refer to products fed to animals when raising livestock. The terms "feed" and "animal feed" are used interchangeably herein.

As used herein, "feed additive" refers to one or more ingredients, substances (e.g., cells) used alone or together in nutrition, for example to improve the quality of food (e.g., animal feed), improve the performance and/or health of animals and/or enhance the digestibility of food or substances in food.

As used herein, the term "food" is used in a broad sense - and encompasses any form of food and food products for humans as well as food (ie, feed) for animals.

The food or feed may be in solution form or in solid form, depending on the use and/or mode of application and/or mode of administration. In some embodiments, the enzymes mentioned herein may be used as food or feed material or in the preparation or production of food or feed material.

As used herein, the term "food or feed ingredient" includes preparations that are added or can be added to food or foods, and includes preparations that can be used at low levels in various products. The food ingredient can be in solution form or solid form, depending on the use and/or application mode and/or administration mode. The enzymes described herein can be used as food or feed ingredients or for preparation or production. These enzymes may or may not be added to food supplements. Feed compositions for monogastric animals generally include compositions comprising plant products that contain phytates. Such compositions include, but are not limited to, corn meal, soybean meal, rapeseed meal, cottonseed meal, corn, wheat, barley, and sorghum-based feeds.

As used herein, the term "granulation" refers to pellets, which may be solid, round, spherical and cylindrical tablets, particularly the production of feed pellets and solid extruded animal feeds. An example of a known feed pelleting manufacturing process generally includes mixing the food or feed ingredients together at room temperature for at least 1 minute, transferring the mixture to a surge bin, conveying the mixture to a steam conditioner (i.e., conditioning), optionally transferring the steam-conditioned mixture to an expander, transferring the mixture to a pelletizer or extruder, and finally transferring the pellets to a pellet cooler. (Fairfield, D. 1994. Chapter 10, Pelleting Cost Center. In Feed Manufacturing Technology IV. (McEllhiney, ed.), American Feed Industry Association, Arlington, Va., pp. 110-139).

The term "pellet" refers to a composition of animal feed (usually derived from cereals) that has been subjected to a heat treatment, such as steam treatment (i.e., conditioning), and pressed or extruded by a machine. The pellets may be incorporated with enzymes in the form of liquid formulations or dry formulations. Dry formulations may be coated or uncoated and may be in the form of granules. The term "granule" is used for particles consisting of an enzyme (e.g., a phytase, such as any of the engineered phytase polypeptides disclosed herein) and other chemicals (e.g., salts and sugars), and may be formed using any of a variety of techniques, including fluidized bed granulation methods to form layered granules.

The term "feed pellet recovery", "recovered activity" or "activity recovery" refers to the ratio of (i) the activity of a feed enzyme after a treatment involving one or more of the following stressors: heating, pressurization, increased pH, decreased pH, storage, drying, exposure to one or more surfactants, exposure to one or more solvents, and mechanical stress to (ii) the activity of the enzyme before the treatment. The recovered activity can be expressed as a percentage. The percentage of recovered activity is calculated as follows:

The phytase may be characterized as stable by showing any of improved "feed pelleting recovery", "recovery activity", "thermal stability" or "inactivity reversibility".

In the context of pelleting experiments, the "activity before treatment" can be approximated by measuring the activity of the phytase present in the untreated mash in a manner that matches the phytase that has been treated. For example, the phytase in the untreated mash is treated and stored for similar times and conditions to the phytase in the treated mash to control for possible interactions or other effects that may be outside the specified treatment itself.

The terms "feed pelleting recovery test" and "standard feed pelleting test" are used interchangeably herein and refer to a test that measures or assesses the stability of a feed enzyme to the heat treatment of conditioning and pelleting.

For example, such a feed pelletizing recovery test is presented in Example 5 below.

The term phytase activity in relation to a determination in a solid or liquid formulation means 1 FTU (phytase unit) which is defined as the amount of enzyme required to liberate 1 micromole of inorganic orthophosphate from 5.0 mM sodium phytate substrate (from rice) within one minute under reaction conditions of 37°C, pH 5.5, as also defined in the ISO 2009 phytase assay (a standard assay for determining phytase activity, see International Standard ISO/DIS 30024:1-17, 2009).

Alternatively, as used herein, one unit of phytase (U) can be defined as the amount of enzyme that releases 1 micromole of inorganic orthophosphate from 0.2 mM sodium phytate substrate (from rice) within one minute under the reaction conditions in the malachite green assay as shown in Example 3 (25°C, pH 5.5 or 3.5, respectively).

As used herein, the term "specific activity" is the number of enzyme units per milliliter (ml) divided by the (total) protein concentration in mg/ml. Therefore, specific activity values are usually expressed in units/mg. Alternatively, the term "specific activity" is the number of enzyme units per milliliter (ml) divided by the phytase concentration in mg/ml.

As used herein, the term "differential scanning calorimetry" or "DSC" is a thermal analysis technique in which the difference in the amount of heat required to increase the temperature of a sample and a reference is measured as a function of temperature. Both the sample and the reference are maintained at nearly the same temperature throughout the experiment. Typically, the temperature program for a DSC analysis is designed so that the sample holder temperature increases linearly as a function of time. The reference sample should have a well-defined heat capacity over the temperature range to be scanned.

The term "probiotic" means a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of beneficial bacteria.

As used herein, the term "direct-fed microorganism" ("DFM") is a source of live (viable) microorganisms that, when applied in sufficient quantities, can confer a benefit to its recipient, i.e., a probiotic. A DFM may contain one or more such microorganisms, such as bacterial strains. Classes of DFM include bacillus, lactic acid bacteria, and yeast. Thus, the term DFM encompasses one or more of: direct-fed bacteria, direct-fed yeast, direct-fed yeast, and combinations thereof.

Bacilli are unique, spore-forming, Gram-positive rods. These spores are very stable and can withstand environmental conditions such as heat, moisture, and a range of pH. These spores germinate into active, vegetative cells when ingested by the animal and can be used in meal and pellet diets. Lactic acid bacteria are Gram-positive cocci that produce lactic acid, which has an antagonistic effect on pathogens. Since lactic acid bacteria appear to be somewhat heat-sensitive, they are not used in pellet diets. Types of lactic acid bacteria include Bifidobacterium, Lactobacillus, and Streptococcus.

The terms "probiotics", "probiotic cultures" and "DFM" are used interchangeably herein and define live microorganisms (including, for example, bacteria or yeast) that beneficially affect a host organism (i.e., by conferring one or more demonstrable health benefits such as health, digestion, and/or performance benefits) when, for example, ingested or applied topically in sufficient quantities. Probiotics can improve the microbial balance of one or more mucosal surfaces. For example, the mucosal surface can be the intestine, urinary tract, respiratory tract, or skin. As used herein, the term "probiotic" also encompasses live microorganisms that can stimulate beneficial arms of the immune system and simultaneously reduce inflammatory responses in mucosal surfaces (e.g., the intestine). Although there is no lower or upper limit for probiotic intake, it has been shown that at least 10 ⁶ -10 ¹² , preferably at least 10 ⁶ -10 ¹⁰ , preferably 10 ⁸ -10 ⁹ cfu as a daily dose will be effective in achieving a beneficial health effect in a subject.

As used herein, the term "CFU" means "colony forming unit" and is a measure of viable cells, wherein a colony represents a collection of cells derived from a single progenitor cell.

The term "isolated" means a substance in a form or environment that does not exist in nature, and does not reflect the degree to which the isolate has been purified, but rather indicates separation or separation from a natural form or natural environment. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance that is at least partially removed from one or more or all of the naturally occurring components with which it is naturally associated, including but not limited to any host cell, enzyme, engineered enzyme, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated. The terms "isolated nucleic acid molecule," "isolated polynucleotide," and "isolated nucleic acid fragment" will be used interchangeably and refer to a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a DNA polymer may be composed of one or more fragments of cDNA, genomic DNA, or synthetic DNA.

The terms "purify," "purified," and purification mean to remove unwanted components, material contaminants, admixtures, or imperfections to render substantially pure or clean. For example, purification, as applied to nucleic acids or polypeptides, generally refers to a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, a chromatographic eluate, and/or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces substantially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., weight percentage on a molar basis). In a related sense, a molecule in a composition is enriched when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other specific substance or component in a composition at a relative or absolute concentration greater than that of the starting composition.

The terms "peptide", "protein" and "polypeptide" are used interchangeably herein and refer to polymers of amino acids linked together by peptide bonds. "Protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. Single-letter and 3-letter codes of amino acids defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. Single letter X refers to any of the twenty amino acids. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. Mutations can be named by the single letter code of the parent amino acid, followed by the position number, and then the single letter code of the variant amino acid. For example, mutation of glycine (G) at position 87 to serine (S) is represented as "G087S" or "G87S". When describing a modification, the position following the amino acid listed in brackets indicates a list of amino acid substitutions at that position that are listed by any of the amino acids. For example, 6 (L, I) means that position 6 can be substituted by leucine or isoleucine. Sometimes in a sequence, a slash (/) is used to define a substitution, for example, F/V indicates that either phenylalanine or valine may be present at that position.

The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can participate in the secretion or directional transport of a mature or precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. Signal sequences are generally not present in mature proteins. Typically, after protein transport, the signal sequence is cut from the protein by a signal peptidase.

The term "mature" form of a protein, polypeptide or peptide refers to the functional form of the protein, polypeptide or enzyme without the signal peptide sequence and the propeptide sequence.

With respect to amino acid sequences or nucleic acid sequences, the term "wild-type" indicates that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., protein, amino acid or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant/engineered nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences).

As used herein, with respect to amino acid residue positions, "corresponding to" or "corresponds to" or "corresponds to" refers to the amino acid residue at the recited position in a protein or peptide, or an amino acid residue that is similar, homologous, or identical to the recited residue in a protein or peptide. As used herein, a "corresponding region" generally refers to an analogous position in a related protein or reference protein.

The terms "derived from" and "obtained from" refer not only to proteins produced or producible by a strain of the organism in question, but also to proteins encoded by a DNA sequence isolated from such a strain and produced in a host organism containing such a DNA sequence. In addition, the term refers to proteins encoded by a DNA sequence of synthetic and/or cDNA origin and having the identifying characteristics of the protein in question.

The term "amino acid" refers to the basic chemical building block of a protein or polypeptide. Abbreviations used herein to identify specific amino acids can be found in Table 1.

Table 1. Single-letter and three-letter amino acid abbreviations

Those of ordinary skill in the art will recognize that the amino acid sequences disclosed herein may be modified while retaining the functions associated with the disclosed amino acid sequences. For example, it is well known in the art that genetic alterations that result in the production of chemically equivalent amino acids at a given site but do not affect the functional properties of the encoded protein are common.

The term "codon-optimized," when it refers to the coding region of a gene or nucleic acid molecule for transformation of a variety of hosts, refers to altering the codons in the coding region of a gene or nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.

The term "gene" refers to a nucleic acid molecule that expresses a specific protein, including regulatory sequences before (5' non-coding sequences) and after (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a natural gene, which contains regulatory sequences and coding sequences that are not found together in nature. Therefore, a chimeric gene can contain regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from natural occurrence. "Endogenous gene" refers to a natural gene located in the natural location of the genome of an organism. "Foreign" gene refers to a gene that is not usually found in the host organism, but is introduced into the host organism by gene transfer. Foreign genes can include natural genes or chimeric genes inserted into non-natural organisms. "Transgenic" is a gene introduced into the genome by a transformation procedure.

The term "intron" means any nucleotide sequence within a gene that is removed by RNA splicing during maturation of the final RNA product. The term "intron" refers to a DNA sequence within a gene and the corresponding sequence in the RNA transcript.

The term "coding sequence" refers to a nucleotide sequence that encodes a specific amino acid sequence. "Suitable regulatory sequence" refers to a nucleotide sequence that is located upstream (5' non-coding sequence), internal or downstream (3' non-coding sequence) of a coding sequence and that affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, RNA processing sites, effector binding sites, and stem-loop structures.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid molecule such that the function of one nucleic acid fragment is affected by the other. For example, a promoter is operably linked to a coding sequence when it is able to affect the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in the sense or antisense orientation.

The terms "regulatory sequence" or "control sequence" are used interchangeably herein and refer to a nucleotide sequence segment that is capable of increasing or decreasing the expression of a specific gene in an organism. Examples of regulatory sequences include, but are not limited to, promoters, signal sequences, operators, etc. As described above, regulatory sequences can be operatively linked to a target coding sequence/gene in a sense or antisense orientation.

"Promoter" or "promoter sequence" refers to a regulatory sequence involved in binding RNA polymerase to initiate transcription of a gene. A promoter can be an inducible promoter or a constitutive promoter. A preferred promoter for use in the present invention is Trichoderma reesei cbh1, which is an inducible promoter.

The "3' non-coding sequence" refers to a DNA sequence located downstream of a coding sequence and includes sequences encoding regulatory signals that can affect mRNA processing or gene expression (eg, transcription termination).

As used herein, the term "transformation" refers to the transfer or introduction of a nucleic acid molecule into a host organism. The nucleic acid molecule can be introduced as a linear or circular form of DNA. The nucleic acid molecule can be an autonomously replicating plasmid, or it can be integrated into the genome of a production host. Production hosts containing transforming nucleic acids are referred to as "transformed" or "recombinant" or "transgenic" organisms or "transformants."

The terms "recombinant" and "engineered" refer to the artificial combination of two originally separated nucleic acid sequence fragments, for example, by chemical synthesis or by manipulating separated nucleic acid fragments through genetic engineering techniques. For example, DNA in which one or more fragments or genes from different molecules, from another part of the same molecule or an artificial sequence have been inserted naturally or through laboratory manipulation, resulting in the introduction of new sequences in the gene and subsequently in the organism. The terms "recombinant", "transgenic", "transformed", "engineered", "genetically engineered" and "modified for exogenous gene expression" are used interchangeably herein.

The terms "recombinant construct," "expression construct," "recombinant expression construct," and "expression cassette" are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that do not exist together in nature. For example, a construct may comprise regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a manner different from that which occurs naturally. Such constructs may be used alone or in combination with a vector. If a vector is used, the choice of vector depends on the method that will be used to transform the host cell, as is well known to those skilled in the art. For example, a plasmid vector may be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells. The skilled artisan will also recognize that different independent transformation events may result in different expression levels and patterns (Jones et al., (1985) EMBO J [Journal of the European Molecular Biology Association] 4:2411-2418; De Almeida et al., (1989) Mol Gen Genetics [Molecular and General Genetics] 218:78-86), and therefore typically screen multiple events to obtain strains that display the desired expression levels and patterns. Such screening can be accomplished using standard molecular biology, biochemistry, and other assays, including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblot analysis of protein expression, enzyme or activity assays, and/or phenotypic analysis.

The terms "production host," "host," and "host cell" are used interchangeably herein and refer to any plant, organism, or cell of any plant or organism, whether human or non-human, into which a recombinant construct can be stably or transiently introduced to express a gene. The term encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during reproduction.

The term "percent identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the number of nucleotides or amino acids that match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M. and Griffin, H.G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); Primer [Sequence Analysis Primers] (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Methods for determining identity and similarity are codified in publicly available computer programs.

As used herein, "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity can be determined using standard techniques known in the art. Useful algorithms include the BLAST algorithm (see, Altschul et al., J Mol Biol [Journal of Molecular Biology], 215:403-410, 1990; and Karlin and Altschul, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5873-5787, 1993). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most related sequences in terms of biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res [Nucleic Acids Research], 25:3389-3402, 1997; and Schaffer et al., Nucleic Acids Res [Nucleic Acids Research], 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searches include: neighborhood word threshold = 11; E-value cutoff = 10; scoring matrix = NUC.3.1 (match = 1, mismatch = -3); gap opening = 5; and gap extension = 2. Exemplary default BLAST parameters for amino acid sequence searches include: word length = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap opening = 11; and gap extension = 1. Amino acid sequence identity percentage (%) values are determined by dividing the number of identical residues matched by the total number of residues in the "reference" sequence. The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

As used herein, "homologous proteins" or "homologous phytases" refer to proteins that have significant similarities in primary, secondary and/or tertiary structure. When aligning proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST using a threshold (E-value cutoff) of 0.001. (Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI BLAST a new generation of protein database search programs. Nucleic Acids Res 1997 Group 1; 25(17): 3389-402). Using this information, protein sequences can be grouped.

版本10.2.4的MAFFT比对以默认设置、评分矩阵BLOSUM62、空位开放罚分1.53和偏移值0.123来导出多序列比对。Sequence alignments and calculations of percent identity can be performed using the Megalign program of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, WI), the AlignX program of Vector NTI v.7.0 (Informax, Inc., Bethesda, MD), or the EMBOSS open source software package (EMBL-EBI; Rice et al., Trends in Genetics 16, (6): 276-277 (2000)). Multiple alignments of sequences can be performed using the CLUSTAL method of alignment (such as CLUSTALW; e.g., version 1.83) (Higgins and Sharp, CABIOS [Bioinformatics], 5:151-153 (1989); Higgins et al., Nucleic Acids Res. [Nucleic Acids Research] 22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res [Nucleic Acids Research] 31(13):3497-500 (2003), obtained from the European Molecular Biology Laboratory via the European Bioinformatics Institute) with default parameters. Suitable parameters for CLUSTALW protein alignments include gap existence penalty=15, gap extension=0.2, matrix=Gonnet (e.g., Gonnet250), protein ENDGAP=-1, protein GAPDIST=4, and KTUPLE=1. In one embodiment, fast or slow alignments use the default settings in the case of slow alignments. Alternatively, the parameters for using the CLUSTALW method (e.g., version 1.83) can be modified to also use KTUPLE=1, gap penalty=10, gap extension=1, matrix=BLOSUM (e.g., BLOSUM64), window=5, and top diagonal of storage=5. Alternatively, the method from

MAFFT version 10.2.4 was used to derive multiple sequence alignments with default settings, scoring matrix BLOSUM62, gap open penalty of 1.53 and offset value of 0.123.

The MUSCLE program (Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput [MUSCLE: multiple sequence alignment with high accuracy and high throughput] Nucl. Acids Res. [Nucleic Acids Research] (2004) 32(5): 1792-1797) is another example of a multiple sequence alignment algorithm.

Figure 2 depicts a phylogenetic or evolutionary tree showing the relatedness among various phytases, including engineered phytase polypeptides and fragments thereof, based on amino acid sequence similarities and differences.

Another way to identify sequence similarity is to generate a hidden Markov model (HMM). HMM is a probabilistic framework in which observed data (e.g., DNA or amino acid sequences) are modeled according to a series of outputs (or emissions) generated by one of several (hidden) internal states. HMMs are commonly used for statistical analysis of multiple DNA sequence alignments. They can be used to identify genomic features such as ORFs, insertions, deletions, substitutions, and protein domains. HMMs can also be used to identify homology; for example, the widely used Pfam database (Punta et al., 2012) is a database of protein families identified using HMMs. HMMs are much more accurate than BLAST (Basic Local Alignment Search Tool), the main tool for sequence comparison tools first produced in 1990 (Altschul et al., 1990, 1997). Therefore, the polypeptide sequences and fragments of the high Tm phytase clade polypeptides shown in Example 4 were used to generate hidden Markov models (HMMs) to identify sequence similarity.

The term "engineered phytase polypeptide" means a polypeptide that does not occur in nature and that has phytase activity.

Note that a fragment of an engineered phytase polypeptide is a portion or subsequence of an engineered phytase polypeptide that is capable of functioning like the engineered phytase polypeptide, ie, it retains phytase activity.

The term "vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, expression cassettes, and the like.

As used herein, "expression vector" means a DNA construct comprising a DNA sequence that is operably linked to a suitable control sequence capable of achieving DNA expression in a suitable host. Such control sequences may include a promoter that achieves transcription, an optional operator sequence that controls transcription, a sequence encoding a suitable ribosome binding site on mRNA, an enhancer, and sequences that control transcription and translation termination.

As used herein, the term "expression" refers to the production of a functional end product (eg, mRNA or protein) in either precursor or mature form. Expression may also refer to the translation of mRNA into a polypeptide.

The expression of a gene involves the transcription of the gene and the translation of mRNA into a precursor or mature protein. A "mature" protein refers to a polypeptide that has been processed post-translationally; that is, a polypeptide from which any signal sequence, prepeptide, or propeptide present in the primary translation product has been removed. A "precursor" protein refers to the primary product of mRNA translation; that is, the propeptide and propeptide are still present. The propeptide and propeptide can be, but are not limited to, intracellular localization signals. "Stable transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, including both the nuclear and organelle genomes, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus of a host organism or into an organelle containing DNA, resulting in gene expression without integration or stable inheritance.

Thus, in one embodiment, a recombinant construct is described that comprises a regulatory sequence functional in a production host operably linked to a nucleotide sequence encoding an engineered phytase polypeptide and fragments thereof as described herein.

The recombinant construct may include a regulatory sequence that functions in a production host, which is operably connected to a nucleotide sequence encoding any engineered phytase polypeptide described herein and fragments thereof. In addition, the production host is selected from the group consisting of bacteria, fungi, yeast, plants or algae. A preferred production host is the filamentous fungus Trichoderma reesei.

Alternatively, it is possible to use cell-free protein synthesis as described in Chong, Curr Protoc Mol Biol. 2014; 108: 16.30.1-16.30.11.

Also described herein is a method for producing an engineered phytase polypeptide or fragment thereof, the method comprising:

(a) transforming a production host with a recombinant construct described herein; and

(b) culturing the production host of step (a) under conditions such that the engineered phytase polypeptide or fragment thereof is produced.

Optionally, the engineered phytase polypeptide or fragment thereof may be recovered from the production host.

In another aspect, a culture supernatant containing phytase may be obtained by any of the methods disclosed herein.

In another embodiment, a polynucleotide sequence encoding any of the engineered phytase polypeptides or fragments thereof as described herein is described.

The possible initiation control region or the promoter that can be included in the expression vector are numerous and are familiar to those skilled in the art." constitutive promoter " is a promoter that is active under most environments and development conditions. " inducible " or " repressible " promoter refers to promoter that is active under environment or development regulation. In certain embodiments, due to the variation of environmental factors, promoter is inducible or repressible, and these environmental factors include but are not limited to the presence of carbon, nitrogen or other nutrients availability, temperature, pH, osmotic pressure, one or more heavy metals, the concentration of one or more inhibitors, pressure or the combination of aforementioned factors as known in the art. In certain embodiments, inducible or repressible promoter can be induced or repressed by metabolic factors, such as the level of some carbon sources as known in the art, the level of some energy sources, the level of some catabolites or the combination of aforementioned factors.

In one embodiment, the promoter is a native promoter of the host cell. For example, in some cases, when Trichoderma reesei is the host, the promoter can be a native Trichoderma reesei promoter, such as the cbhl promoter deposited in GenBank under Accession No. D86235. Other suitable non-limiting examples of promoters that can be used for fungal expression include cbh2, egl1, egl2, egl3, egl4, egl5, xyn1 and xyn2, the repressible acid phosphatase gene (phoA) promoter of Penicillium chrysogenus (P. chrysogenus) (see, e.g., Graessle et al., (1997) Appl. Environ. Microbiol., 63:753-756), the glucose-repressible PCK1 promoter (see, e.g., Leuker et al., (1997), Gene, 192:235-240), the maltose-inducible, glucose-repressible MET3 promoter (see Liu et al., (2006), Eukary. Cell, 5:638-649), the pKi promoter and the cpc1 promoter. Other examples of useful promoters include promoters from A. awamori and A. niger glucoamylase genes (see, e.g., Nunberg et al., (1984) Mol. Cell Biol. 15 4: 2306-2315 and Boel et al., (1984) EMBO J. 3: 1581-1585). In addition, the promoter of the Trichoderma reesei xln1 gene may be useful (see, e.g., EPA 137280A1).

The DNA fragment controlling transcription termination can also be derived from various natural genes of preferred production host cells. In certain embodiments, including a termination control region is optional. In certain embodiments, the expression vector includes a termination control region derived from a preferred host cell.

The terms "production host", "production host cell", "host cell" and "host strain" are used interchangeably herein and refer to a suitable host of an expression vector or DNA construct comprising a polynucleotide encoding a phytase polypeptide or fragment thereof. The choice of production host may be selected from the group consisting of bacteria, fungi, yeast, plants and algae. Typically, the choice will depend on the gene encoding the engineered phytase polypeptide or fragment thereof and its source.

In particular, the host strain is preferably a filamentous fungal cell.In a preferred embodiment of the present invention, "host cell" means cells and protoplasts produced by filamentous fungal strains, and in particular cells of Trichoderma species or Aspergillus species.

The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina (see Alexopoulos, C.J. (1962), INTRODUCTORY MYCOLOGY, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically and genetically distinct from yeasts. Vegetative growth of filamentous fungi is by hyphal elongation, and carbon catabolism is obligately aerobic. In the present invention, the filamentous fungal parent cell can be, but is not limited to, a cell of the following species: Trichoderma (e.g., Trichoderma reesei (formerly classified as T. longibrachiatum and currently also known as Hypocrea jecorina), Trichoderma viride, Trichoderma koningii, Trichoderma harzianum); Penicillium species, Humicola species (e.g., Humicola insolens and Humicola grisea) grisea), Chrysosporium species (e.g., C. lucknowense), Gliocladium species, Aspergillus species (e.g., A. oryzae, A. niger, and A. awamori), Fusarium species, Neurospora species, Hypocrea species, and Emericella species (see also Innis et al., (1985) Sci. [Science Citation Index] 228:21-26).

As used herein, the term "Trichoderma" or "Trichoderma species" refers to any genus of fungi previously or currently classified as Trichoderma.

The expression cassette may be included in a production host, particularly a cell of a microbial production host. The production host cell may be a microbial host found in the fungus family, and grows under a wide range of temperature, pH value, and solvent tolerance. For example, any one of an expected bacterium, yeast, plant, algae, or fungus (such as a filamentous fungus) may be suitably used as a host for an expression vector.

Including an expression cassette in a production host cell can be used to express the protein of interest so that it can be located intracellularly, extracellularly, or a combination of both. Extracellular expression makes it easier to recover the desired protein from the fermentation product compared to methods for recovering proteins produced by intracellular expression.

Methods for converting nucleic acids into filamentous fungi (such as Aspergillus spp., for example A. oryzae or A. niger, H. grisea, H. insolens and Trichoderma reesei) are well known in the art. Suitable procedures for transforming Aspergillus host cells are described in, for example, EP 238023.

Suitable procedures for transforming Trichoderma host cells are described, for example, in Steiger et al. 2011, Appl. Environ. Microbiol. 77: 114-121. The uptake of DNA into the host Trichoderma species strain depends on the calcium ion concentration. Typically, about 10 mM CaCl ₂ to 50 mM CaCl ₂ is used in the uptake solution. In addition to the need for calcium ions in the uptake solution, other compounds that are typically included are buffer systems such as TE buffer (10 MmTris, pH 7.4; 1 mM EDTA) or 10 mM MOPS (pH 6.0) buffer (morpholinepropanesulfonic acid) and polyethylene glycol (PEG). It is believed that the polyethylene glycol acts to fuse the cell membrane, thereby allowing the contents of the culture medium to be delivered to the cytoplasm of the Trichoderma species strain and transfer the plasmid DNA to the nucleus. This fusion often leaves multiple copies of the plasmid DNA integrated into the host chromosome.

Suspensions containing Trichoderma species protoplasts or cells that have been subjected to osmotic treatment at a density of 10 ⁵ to 10 ⁷ /mL, preferably 2×10 ⁶ /mL, are typically used in transformation. These protoplasts or cells in a volume of 100 μL are mixed with the desired DNA in an appropriate solution (e.g., 1.2 M sorbitol; 50 mM CaCl ₂ ). Typically, a high concentration of PEG is added to the uptake solution. 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension. However, adding about 0.25 volume to the protoplast suspension is preferred. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc. can also be added to the uptake solution and aid in transformation. Similar procedures can be used for other fungal host cells. (See, e.g., U.S. Patent Nos. 6,022,725 and 6,268,328, both of which are incorporated herein by reference).

Preferably, a genetically stable transformant is constructed using a vector system whereby the nucleic acid encoding the phytase polypeptide or fragment thereof is stably integrated into the host strain chromosome. The transformant is then purified by known techniques.

After the expression vector has been introduced into the cells, the transfected or transformed cells are cultured under conditions that favor expression of the gene under the control of the promoter sequence.

Typically, cells are cultured in standard culture media containing physiological salts and nutrients (see, e.g., Pourquie, J. et al., BIOCHEMISTRY AND GENETICS OF CELLULOSE DEGRADATION, Aubert, J.P. et al., eds., Academic Press, pp. 71-86, 1988 and IImen, M. et al., (1997) Appl. Environ. Microbiol. 63: 1298-1306). Common commercially prepared culture media (e.g., yeast malt extract (YM) broth, Luria-Bertani (LB) broth, and Sabouraud Dextrose (SD) broth can also be used in the present invention.

Cultivation conditions are also standard (e.g., incubating the culture at about 28° C. in a suitable medium in a shaking culture or fermenter until the desired phytase expression level is reached). Preferred culture conditions for a given filamentous fungus are known in the art and can be found in the scientific literature and/or from sources of fungi such as the American Type Culture Collection and the Fungal Genetics Stock Center.

After fungal growth has been established, the cells are exposed to conditions effective to cause or allow expression of phytase, and in particular phytase as defined herein. In the case where the phytase coding sequence is under the control of an inducible promoter, an inducer (e.g., a sugar, a metal salt, or an antimicrobial agent) is added to the culture medium at a concentration effective to induce expression of phytase. Engineered phytase polypeptides or fragments thereof secreted from the host cells can be used as a whole broth formulation with minimal post-production processing.

Preparation of spent whole fermentation broth of a recombinant microorganism resulting in expression of an engineered phytase polypeptide or fragment thereof can be accomplished using any culture method known in the art.

The term "spent whole fermentation broth" is defined herein as the unfractionated contents of the fermentation material including culture medium, extracellular proteins (e.g., enzymes), and cell biomass. It should be understood that the term "spent whole fermentation broth" also encompasses cell biomass that has been lysed or permeabilized using methods well known in the art.

After fermentation, a fermentation broth is obtained, and the microbial cells and various suspended solids (including residual crude fermentation material) are removed by conventional separation techniques to obtain a phytase solution. Typically, filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, ultrafiltration after centrifugation, extraction or chromatography are used.

It is possible to optionally recover the desired protein from the production host. In another aspect, the engineered phytase polypeptide or fragment thereof is obtained by using any method known to those skilled in the art containing culture supernatant.

Examples of such techniques include, but are not limited to, affinity chromatography (Tilbeurgh et al., (1984) FEBS Lett. 16:215), ion exchange chromatography (Goyal et al., (1991) Biores. Technol. 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol. 17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. 6:336; and Ellouz et al., (1987) Chromatography 396:307), including ion exchange using materials having high resolution capabilities (Medve et al., (1998) J. Chromatography 396:307). A [Journal of Chromatography A] 808: 153), hydrophobic interaction chromatography (see, Tomaz and Queiroz, (1999) J. Chromatography A [Journal of Chromatography A] 865: 123); two-phase partitioning (see, Brumbauer et al., (1999) Bioseparation [Biological Separation] 7: 287); ethanol precipitation; reverse phase HPLC, silica gel or cation exchange resin chromatography such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration (e.g., Sephadex G-75). The desired degree of purification will vary depending on the use of the engineered phytase polypeptide or fragment thereof. In some embodiments, purification will not be necessary.

On the other hand, it may be necessary to concentrate the solution containing the engineered phytase polypeptide or its fragment in order to optimize the recovery. Using unconcentrated solution requires increasing the incubation time in order to collect the enzyme precipitate through enrichment or purification. Use conventional concentration techniques to concentrate the enzyme-containing solution until the desired enzyme level is obtained. The concentration of the enzyme-containing solution can be achieved by any of the techniques discussed in this article. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and/or ultrafiltration.

In addition, the concentration of the desired protein product can be carried out using, for example, a precipitant such as a metal halide precipitant. The metal halide precipitant sodium chloride can also be used as a preservative. The metal halide precipitant is used in an amount that effectively precipitates the engineered phytase polypeptide or its fragment. After routine testing, it will be apparent to those of ordinary skill in the art to select at least an effective amount and an optimal amount of metal halide to effectively cause precipitation of the enzyme, as well as precipitation conditions for maximizing recovery, including incubation time, pH, temperature and enzyme concentration. Typically, at least about 5% w/v (weight/volume) to about 25% w/v of metal halide is added to the concentrated enzyme solution, and typically at least 8% w/v.

Another alternative to precipitating enzymes is to use organic compounds. Exemplary organic compound precipitation agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of organic compound precipitation agents can be carried out before, simultaneously with, or after the addition of metal halide precipitation agents, and the addition of two precipitation agents, organic compounds, and metal halides can be carried out sequentially or simultaneously. Typically, organic precipitation agents are selected from the group consisting of: alkali metal salts of 4-hydroxybenzoic acid (such as sodium salts or potassium salts), and straight or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains 1 to 12 carbon atoms, and blends of two or more of these organic compounds. Other organic compounds also include, but are not limited to, methyl 4-hydroxybenzoate (called methyl p-hydroxybenzoate), propyl 4-hydroxybenzoate (called propyl p-hydroxybenzoate). For further description, see, for example, U.S. Patent No. 5,281,526. The addition of an organic compound precipitation agent provides the advantage of high flexibility in precipitation conditions in terms of pH, temperature, concentration, precipitation agent, protein concentration and incubation time. Typically, at least about 0.01% w/v and no more than about 0.3% w/v of an organic compound precipitation agent is added to the concentrated enzyme solution.

After the incubation period, the enzyme of enrichment or purification is then separated from the pigment and other impurities of dissociation, and collected by conventional separation techniques such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, filter press, cross-membrane microfiltration, cross-flow membrane microfiltration, etc. The further enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water. For example, the enzyme precipitate of enrichment or purification can be obtained by washing with water containing a metal halide precipitation agent or with a water washing containing a metal halide and an organic compound precipitation agent.

Sometimes it is advantageous to delete genes from an expression host, where the gene defect can be cured by the expression vector. Where it is desired to obtain a fungal host cell with one or more inactivated genes, known methods (e.g., methods disclosed in U.S. Pat. No. 5,246,853, U.S. Pat. No. 5,475,101, and WO 92/06209) may be used. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation, or by any other means that renders the gene inoperative for its intended purpose (such that the gene is prevented from expressing a functional protein).

Any cloned gene from Trichoderma species or other filamentous fungal hosts can be deleted, such as cbh1, cbh2, egl1 and egl2 genes. In some embodiments, gene deletion can be completed by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art. Then, within the desired gene coding region, the deletion plasmid is cut at one or more appropriate restriction enzyme sites, and the gene coding sequence or part thereof is replaced with a selective marker. Flanking DNA sequences (preferably between about 0.5 and 2.0 kb) from the locus of the gene to be deleted are retained on either side of the marker gene. Appropriate deletion plasmids will generally have unique restriction enzyme sites present therein so that the fragment containing the deleted gene (including the flanking DNA sequences) and the selective marker gene can be removed as a single linear piece.

Depending on the host cell used, post-transcriptional and/or post-translational modifications may be performed. A non-limiting example of post-transcriptional and/or post-translational modifications is "chopping" or "truncating" of the polypeptide. In another case, such chopping may result in obtaining a mature phytase polypeptide and further removing the N or C-terminal amino acid to produce a truncated form of the phytase that retains enzymatic activity.

Other examples of post-transcriptional or post-translational modifications include, but are not limited to, myristoylation, glycosylation, truncation, lipidation, and tyrosine, serine or threonine phosphorylation. Those skilled in the art will recognize that the type of post-transcriptional or post-translational modification that a protein may undergo may depend on the host organism in which the protein is expressed.

Further sequence modifications may occur to the polypeptide after expression. This includes, but is not limited to, oxidation, deglycosylation, saccharification, etc. It is known that saccharification can affect the activity of phytase when incubated with glucose or other reducing sugars, particularly at temperatures above 30°C and neutral or alkaline pH. Protein engineering to eliminate lysine residues can be used to prevent such modifications. Such examples can be found in US 8,507,240. For example, yeast expression can produce highly glycosylated polypeptides, resulting in a significant increase in molecular weight. In addition, WO2013/119470, with an international publication date of August 15, 2013 (incorporated herein by reference), relates to phytases with increased stability (which is believed to be due to increased glycosylation).

As used herein, the term "glycosylation" refers to the attachment of a polysaccharide to a molecule, such as a protein. Glycosylation can be an enzymatic reaction. Attachment can be formed by a covalent bond. The phrase "highly glycosylated" refers to a molecule (such as an enzyme) being glycosylated at many sites and all or nearly all available glycosylation sites (e.g., N-linked glycosylation sites). Alternatively or in addition thereto, the phrase "highly glycosylated" can refer to a wide range of glycolytic branches (such as, the size and number of glycolytic moieties associated with a specific N-linked glycosylation site) at all or substantially all N-linked glycosylation sites. In some embodiments, the engineered phytase polypeptide is glycosylated at all or substantially all consensus sequence N-linked glycosylation sites (i.e., NXS/T consensus sequence N-linked glycosylation sites).

As used herein, the term "glycan" refers to a polysaccharide or oligosaccharide, or the carbohydrate portion of a glycoconjugate (such as a glycoprotein). Glycans can be homopolymers or heteropolymers of monosaccharide residues. They can be straight-chain or branched molecules.

Phytases may have varying degrees of glycosylation. Such glycosylation is known to increase stability during storage and application.

The activity of any of the engineered phytase polypeptides or fragments thereof disclosed herein can be determined as discussed above.

As will be appreciated by those skilled in the art, enzymes are fragile proteins that are always threatened in the harsh environment of a feed mill. Extreme temperatures, pressures, friction, pH, and microbial activity can degrade or destroy enzymes added to the feed. Enzyme activity stress occurs primarily during the conditioning and pelleting stages of processing. For example, the feed absorbs most of its thermal energy during conditioning before pelleting. However, passing the pellet die from the conditioner can also heat the feed. Many factors may cause the temperature to rise through the die, such as feed formulation, die thickness, die speed, die specifications (hole size and shape), initial processing temperature, pelleting capacity, etc.

-og

)，食品局(

)，编号2017-32-31-00378)。Therefore, conditions during pelleting of feed on an industrial scale can vary. The ability or robustness of the enzyme to withstand these variations under pelleting conditions is very important. One of ordinary skill in the art will appreciate that the regulating temperature can vary depending on the feed mill. In addition, local laws need to be considered when determining the conditions under which the pelleting process is to be performed. For example, Danish law requires that pelleting of poultry feed be set at 81°C (Ministry of Environment and Food (

-og

), Food Bureau (

), No. 2017-32-31-00378).

Likewise, higher temperature pelleting conditions can be used in the industry to improve pellet quality (e.g. better durability and reduced fines) and increase pellet compressibility. Therefore, there is a need for a robust phytase that can produce stable active pellets over a wide temperature range above 80°C when incorporated into the feed prior to conditioning and pelleting. This is important for both liquid applied phytases and solid applied phytases as described herein.

Factors beyond conditioning temperature that may affect the actual stress that feed enzymes may be subjected to include, but are not limited to, feed ingredients, geographic location of the feed mill, equipment used, die size, use of pelleting aids, steam control, temperature control, and any other commercially relevant pelleting conditions, such as the presence of any other exogenous enzymes that have been modified in such a way as to reduce pelleting stress.

These stress factors are further complicated by the tendency towards high temperatures or super conditioning, which results in the application of enzymes in liquid form after pelleting.

What if robust enzymes could be engineered so they could be applied as liquids prior to conditioning and pelleting?

The terms "robust" and "robustness" are used interchangeably herein and refer to the ability of the engineered phytase disclosed herein or fragments thereof to withstand the variations in the conditioning and pelleting processes in industrial feed production. As part of the high Tm-phytase clade polypeptides and fragments thereof, the engineered phytase polypeptides and fragments thereof disclosed herein are examples of robust enzymes that can be applied to feed in liquid form prior to conditioning and pelleting.

In other words, the novel engineered phytase polypeptides and fragments thereof are able to withstand such changes in an unformulated, uncoated, unprotected form during industrial feed pelleting when applied in liquid form prior to conditioning and pelleting or when applied in an unformulated, uncoated, unprotected solid form.

The terms "liquid", "liquid form" and "liquid formulation" are used interchangeably and mean any way in which the enzyme can be applied to the feed in liquid form prior to conditioning and pelleting.

It is believed that applying a robust engineered phytase polypeptide or fragment thereof to the feed in liquid form is beneficial compared to applying such phytase in the form of coated granules. Such coated granules are the current commercial method for making phytase products suitable for high temperature conditioning and pelleting. The benefits of liquid application of the robust enzyme include: 1) the enzyme will begin to work immediately after ingestion by the animal because the enzyme does not have to be released from the coated granules before interacting with the feed, 2) the distribution of the enzyme throughout the feed is improved, thus ensuring a more consistent delivery of the enzyme to the animal, which is particularly important for young animals that eat small amounts of feed, 3) uniform distribution in the feed makes it easier to measure the enzyme in the feed, and 4) in the case of a robust phytase (such as the engineered phytase polypeptides and fragments disclosed herein), it can begin to degrade phytate already present in the feed.

In other words, the novel engineered phytase polypeptides and fragments thereof are so robust that no special coatings or formulations are required to apply them to feed prior to conditioning and pelleting because they have been engineered to withstand the stresses of conditioning and pelleting used in industrial feed production. Thus, the robustness of the novel engineered phytase polypeptides and fragments thereof described herein allows them to be applied in the form of uncoated granules or particles or in an uncoated and unprotected form when placed in a liquid.

It should be noted that the engineered phytase polypeptides and fragments thereof can be formulated inexpensively on solid carriers without particular need for protective coatings and remain active throughout the conditioning and pelleting process. A protective coating providing additional thermal stability when applied in solid form may be beneficial for obtaining pelleting stability when required in certain areas where harsh conditions are used or if conditions permit (e.g. in the case of super-conditioned feeds above 90°C).

The disclosed engineered phytase polypeptides or fragments thereof are derived using a combination of methods and techniques known in the art of protein engineering, including phylogenetic analysis, site-evaluated libraries, combinatorial libraries, high throughput screening, and statistical analysis.

In one aspect, the present disclosure relates to an engineered phytase polypeptide or fragment thereof that also has at least 82% sequence identity to the amino acid sequence of SEQ ID NO: 1.

One skilled in the art will understand that such at least 82% sequence identity also includes 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

One skilled in the art will understand that such at least 79% sequence identity also includes 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

The following may also be mentioned, as in some embodiments, it is provided:

a) an engineered phytase polypeptide or fragment thereof that further has at least 81% (such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 2, 3, 8, 10, 12, 18, 19, 24, 26, 27, 28, 30, 31, 32, 33, and/or 36.

b) an engineered phytase polypeptide or fragment thereof that further has at least 82% (such as 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 1, 4, 5, 7, 9, 11, 14, 15, 17, 21, 25, 34, and/or 35;

c) an engineered phytase polypeptide or fragment thereof that also has at least 83% (such as 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 13;

d) an engineered phytase polypeptide or fragment thereof, which further has at least 79% (e.g., 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 6, 22, and/or 64; and/or

e) an engineered phytase polypeptide or fragment thereof that further has at least 80% (such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 16, 20, 23, 29, and/or 37.

In a further aspect, the polypeptide comprises the core domain of the engineered phytase polypeptide or is a core domain fragment of the engineered phytase polypeptide. "Core domain fragment" is defined herein as a polypeptide that lacks one or more amino acids from the amino and/or carboxyl termini of the polypeptide. As used herein, the phrase "core domain" refers to a polypeptide region that encompasses the amino acids necessary for maintaining the structure and function of the polypeptide (such as phytic acid hydrolysis). The amino acids in the core domain may be further modified to improve thermal stability or catalytic activity under various conditions (such as, but not limited to, pH). In some non-limiting embodiments, the core domain of the engineered phytase polypeptide disclosed herein or its fragment corresponds to amino acid positions 14-325 of SEQ ID NO:1. In other non-limiting embodiments, the core domain corresponds to amino acid positions 13-326, 12-327, 11-328, 10-329, 9-330, 8-331, 7-332, 6-333, 5-334, 4-335, 3-336, 2-337, or 1-338 of SEQ ID NO: 1. In other embodiments, the N-terminus of the core domain corresponds to amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 of SEQ ID NO: 1, and the C-terminus of the core domain corresponds to SEQ ID NO: Amino acid position 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363 404, 405, 406, 407, 408, 409, 410, 411, 412, or 413.

Therefore, this article also provides:

f) an engineered phytase polypeptide or a core domain fragment thereof having at least 78% (e.g., 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO: 6 and/or 64, wherein the amino acid positions correspond to amino acid positions of SEQ ID NO: 1;

g) an engineered phytase polypeptide or a core domain fragment thereof having at least 79% (such as 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO: 2, 8, 27 and/or 37, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1;

h) an engineered phytase polypeptide or a core domain fragment thereof having at least 81% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO:3, 10, 12, 18, 25, 26, 28, 30, 32, 35, 65, 70 and/or 86, wherein the amino acid positions correspond to amino acid positions of SEQ ID NO: 1;

i) an engineered phytase polypeptide or a core domain fragment thereof having at least 82% (e.g., 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO: 1, 4, 5, 7, 9, 11, 13-17, 21, 22, 31, 33, 34, 36, 64, 66-69 and/or 71-84, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1;

j) an engineered phytase polypeptide or a core domain fragment thereof having at least 83% (such as 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO: 19, 20, 23 and/or 24, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1; and/or

k) an engineered phytase polypeptide or a core domain fragment thereof having at least 84% (such as 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to amino acids 14-325 of SEQ ID NO:29, wherein the amino acid positions correspond to the amino acid positions of SEQ ID NO:1.

In still another aspect, the engineered phytase polypeptide or fragment thereof having at least 82% sequence identity to the amino acid sequence of SEQ ID NO: 1 can also have an amino acid sequence having a Hidden Markov Model (HMM) score of at least about 1200 (e.g., at least about 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750 or 1800), as shown in Table 11 for high Tm phytase clade polypeptides.

In still other aspects, any engineered phytase polypeptide disclosed herein or its fragment may comprise one or more specific amino acid substitutions at one or more positions within its polypeptide sequence. Similarly, in some embodiments, provided herein are engineered phytase polypeptides or its fragments comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) amino acid substitutions selected from the group consisting of 30 (L, I), 37Y, 45P, 89T, 182R, 194M, 195F, 202S, 228Y, 256H, 261H, and 298V, wherein the position corresponds to the numbering of SEQ ID NO: 1 or 57.

Alternatively or additionally, any of the engineered phytase polypeptides or fragments thereof may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) amino acid substitutions selected from the group consisting of 3T, 6S, 9Q, 73I, 76K, 78S, 118Q, 123A, 130V, 163P, 186D, 187K, 209A, 284S, 288A, 289R, 337V, 345A, and 347K, wherein the positions correspond to the numbering of SEQ ID NO: 1. In some embodiments, the engineered phytase polypeptide is selected from the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87.

The engineered phytase polypeptides or fragments thereof containing one or more amino acid substitutions may exhibit one or more improved or enhanced properties, such as, but not limited to, improved thermostability (e.g., an improvement in thermostability of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more, including all percentages between these values), or improved activity (e.g., an improvement in activity at pH 5.5 compared to activity at pH 5.5). 3.5) (e.g., an improvement in activity of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more (including all percentages between these values)).

In yet other aspects, any of the engineered phytase polypeptides disclosed herein or fragments thereof may have one or more substitutions (e.g., one or more of the substitutions disclosed above) that increase the ratio of the activity (e.g., specific activity) of the phytase at pH 3.5 to that at pH 5.5. Thus, in some embodiments, the ratio of the activity (e.g., specific activity) of any of the engineered phytase polypeptides disclosed herein or fragments thereof at pH 3.5 to that at pH 5.5 is greater than or equal to about 1.2 (e.g., any of about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 or more).

Also described is an engineered phytase polypeptide or fragment thereof having a feed pelleting recovery of at least 50% when applied in MLA at 95°C for 30 seconds using a standard feed pelleting recovery test. In addition, the engineered phytase polypeptide or fragment thereof having a feed pelleting recovery of at least 50% as described herein may also have a sequence identity of at least 82% to the amino acid sequence shown in SEQ ID NO: 1.

The feed pelletizing recovery rate can be any value within the range of from about 50% to about 100%, specifically about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In some embodiments, the engineered phytase polypeptide or fragment thereof has a feed pelleting recovery of at least about 60%, 65%, 70%, 75%, 70%, 85%, 90%, 95% or 99% when applied as a solid at 95°C.

Those skilled in the art will appreciate that feed pelleting recovery may vary depending on the type of feed used, the conditioning and pelleting conditions used (e.g., temperature and moisture content, assays used to determine activity, etc.).

Using a standard feed pelleting test, any of the engineered phytase polypeptides or fragments thereof disclosed herein have a feed pelleting recovery ratio of at least 0.7 when applied in MLA at 95°C for 30 seconds compared to 80°C for 30 seconds. This ratio includes about 0.7, 0.8, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 and 0.99.

In another embodiment, an engineered phytase polypeptide or fragment thereof is disclosed, wherein the ratio of feed pelleting recovery when used in MLA at 95°C for 30 seconds compared to MLA at 80°C for 30 seconds is at least about 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 compared to: a) SEQ ID NO:60; b) SEQ ID NO:60 with A25F and G157R substitutions; c) SEQ ID NO:104; and/or d) amino acids 22-431 of SEQ ID NO:104.

In another embodiment, an engineered phytase polypeptide or fragment thereof is disclosed, wherein the ratio of the feed pelleting recovery when used in MLA at 95°C for 30 seconds compared to MLA at 80°C for 30 seconds is at least about 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 compared to: a) SEQ ID NO:60; b) SEQ ID NO:60 with A25F and G157R substitutions; c) SEQ ID NO:104; and/or d) amino acids 22-431 of SEQ ID NO:104.

Any of the engineered phytase polypeptides or fragments thereof disclosed herein may further comprise a Tm temperature of at least about 92.5°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, about 100°C, or about 101°C (using the differential _{scanning calorimetry} conditions described in Example 3), and the results are provided in Example 4.

In another embodiment, an engineered phytase polypeptide or fragment thereof is disclosed, having a Tm temperature ratio of at least about 1.08, 1.10, 1.12, 1.14, 1.16, 1.18 or 1.20, 2.1, 2.4, 2.7, 3.0 or 3.3 (as measured by differential scanning calorimetry) compared to: a) SEQ ID NO: 60; b) SEQ ID NO: 60 having A25F and G157R substitutions; c) SEQ ID NO: 104; and/or d) amino acids 22-431 of SEQ ID NO: 104.

In another aspect, any of the engineered polypeptides disclosed herein or fragments thereof comprise a specific activity of at least about 100 U/mg at pH 3.5 under assay conditions such as those described in Example 4. Ranges of specific activity (U/mg at pH 3.5) include, but are not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.

On the other hand, some engineered polypeptides or fragments thereof disclosed herein comprise a specific activity of at least about 100 U/mg at pH 5.5 under assay conditions such as those described in Example 4. Ranges of specific activity (U/mg at pH 5.5) include, but are not limited to, about 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 2000, etc.

In still another aspect, any of the engineered phytase polypeptides disclosed herein or fragments thereof may be stable in liquid form at a pH of about 3.0 or lower. This is particularly relevant when the engineered phytase polypeptides described herein or fragments thereof pass through the digestive tract of an animal, as discussed below.

In another embodiment, an animal feed, feedstuff, feed additive composition or premix comprising any of the engineered phytase polypeptides or fragments thereof described herein is described.

Importantly, feed additive enzymes (e.g., phytase) are subject to very harsh conditions when passing through the animal digestive tract, i.e., the presence of low pH and digestive enzymes. Pepsin is one of the most important proteolytic digestive enzymes present in the gastrointestinal tract of monogastric animals. Pepsin has low specificity and high pH tolerance in acidic regions (pH 1.5-6.0 is stable until pH 8.0). The engineered phytase polypeptides described herein or fragments thereof tolerate pepsin to a great extent, and pepsin is essential for good in vivo performance.

Animal feed, feedstuff, feed additive composition or premix comprising any engineered phytase polypeptide or fragment thereof described herein can be used (i) alone, or (ii) in combination with a direct-fed microbial comprising at least one bacterial strain, or (iii) in combination with at least one other enzyme, or (iv) in combination with a direct-fed microbial comprising at least one bacterial strain and at least one other enzyme, or (v) any of (i), (ii), (iii) or (iv), further comprising at least one other feed additive component, and optionally the engineered phytase polypeptide or fragment thereof is present in an amount of at least 0.1 g/ton of feed.

The terms "feed additive", "feed additive component" and/or "feed additive ingredient" are used interchangeably herein.

Feed additives can be described as products used in animal nutrition with the aim of improving feed quality and the quality of animal-derived food, or improving animal performance and health, for example by providing enhanced digestibility of feed materials.

Feed additives fall into a variety of categories, such as sensory additives, which stimulate the animal's appetite, making them naturally want to eat more. Nutritional additives provide specific nutrients that may be lacking in the animal's diet. Animal husbandry additives improve the overall nutritional value of the animal's diet through additives in the feed.

Examples of such feed additives include, but are not limited to, probiotics, essential oils (such as, but not limited to, thymol and/or cinnamaldehyde), fatty acids, short-chain fatty acids (such as propionic acid and butyric acid, etc.), vitamins, minerals, amino acids, etc.

The feed additive composition or formulation may further comprise at least one component selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben and propylparaben.

At least one other enzyme (i.e., in addition to any of the engineered phytase polypeptides or fragments thereof disclosed herein) may be included in the feed additive composition or formulation disclosed herein, which may include, but is not limited to, a xylanase, an amylase, another phytase, a β-glucanase, and/or a protease.

Xylanase is the name of a class of enzymes that degrade the linear polysaccharide β-1,4-xylan to xylose, thereby breaking down hemicellulose, one of the main components of plant cell walls. Xylanases, such as endo-β-xylanase (EC 3.2.1.8), hydrolyze the xylan backbone.

In one embodiment, the xylanase can be any commercially available xylanase. Suitably, the xylanase can be an endo-1,4-P-d-xylanase (classified as E.G.3.2.1.8) or a 1,4β-xylosidase (E.G.3.2.1.37). In one embodiment, the present disclosure relates to a composition comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof in combination with an endo-xylanase (e.g., an endo-1,4-P-d-xylanase) and another enzyme. All E.C. enzyme classifications mentioned herein relate to the classification provided in the Enzyme Nomenclature—Recommendations (1992)—ISBN 0-12-226164-3 of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, which is incorporated herein by reference.

或Avizyme

中的木聚糖酶，两者都是可从丹尼斯科公司(Danisco A/S)商购获得的产品。在一个实施例中，木聚糖酶可以是两种或更多种木聚糖酶的混合物。在另一个实施例中，木聚糖酶是内切-1,4-β-木聚糖酶或1,4-β-木糖苷酶。In another embodiment, the xylanase may be a xylanase from Bacillus, Trichodermna, Therinomyces, Aspergillus, Humicola, and Penicillium. In another embodiment, the xylanase may be a xylanase from Axtra

or Avizyme

In one embodiment, the xylanase may be a mixture of two or more xylanases. In another embodiment, the xylanase is an endo-1,4-β-xylanase or a 1,4-β-xylosidase.

In one embodiment, the disclosure relates to a composition comprising any of the engineered phytase polypeptides disclosed herein or fragments thereof and a xylanase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750 and greater than 750 xylanase units/g composition.

In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, and greater than 8000 xylanase units/g composition.

It is understood that one xylanase unit (XU) is the amount of enzyme which releases 0.5 μmol reducing sugar equivalents (calculated as xylose by the dinitrosalicylic acid (DNS) assay - reducing sugar method) per minute from oat-spelt-xylan substrate at pH 5.3 and 50° C. (Bailey et al., Journal of Biotechnology, Vol. 23, (3), May 1992, 257-270).

Amylases are a class of enzymes that are able to hydrolyze starch into shorter chain oligosaccharides such as maltose. The glucose moiety can then be transferred from maltose to monoglycerides or glycosyl monoglycerides more easily than from the original starch molecule. The term amylase includes alpha-amylases (E.G.3.2.1.1), G4 forming amylases (E.G.3.2.1.60), beta-amylases (E.G.3.2.1.2) and gamma-amylases (E.C.3.2.1.3). Amylases may be of bacterial or fungal origin, or chemically modified or protein engineered mutants.

或Avizyme

中的α-淀粉酶，两者都是可从丹尼斯科公司商购获得的产品。在另一个实施例中，淀粉酶可以是抗胃蛋白酶α-淀粉酶，诸如抗胃蛋白酶木霉属(诸如里氏木霉)α淀粉酶。在英国申请号101 1513.7(其通过援引并入本文)和PCT/IB2011/053018(其通过援引并入本文)中传授了一种合适的抗胃蛋白酶α-淀粉酶。In one embodiment, the amylase can be a mixture of two or more amylases. In another embodiment, the amylase can be an amylase (e.g., α-amylase) from Bacillus licheniformis and an amylase (e.g., α-amylase) from Bacillus amyloliquefaciens. In one embodiment, the α-amylase can be an amylase (e.g., α-amylase) from Axtra

or Avizyme

In another embodiment, the amylase may be a pepsin-resistant alpha-amylase, such as a pepsin-resistant Trichoderma (such as Trichoderma reesei) alpha-amylase. A suitable pepsin-resistant alpha-amylase is taught in UK Application No. 101 1513.7 (incorporated herein by reference) and PCT/IB2011/053018 (incorporated herein by reference).

It will be appreciated that one amylase unit (AU) is the amount of enzyme that releases 1 mmol of glycosidic bonds per minute from a water-insoluble cross-linked starch polymer substrate at pH 6.5 and 37°C (this may be referred to herein as an assay to determine 1 AU).

In one embodiment, the disclosure relates to a composition comprising any engineered phytase polypeptide disclosed herein or a fragment thereof and an amylase. In one embodiment, the disclosure relates to a composition comprising any engineered phytase polypeptide disclosed herein or a fragment thereof, a xylanase and an amylase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750 and greater than 750 amylase units/g composition.

In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-700 -15,000, 14,000-15,000, and more than 15,000 amylase units/g of composition.

As used herein, the term protease is synonymous with peptidase or proteinase. The protease may be a subtilisin (E.G.3.4.21.62) or a bacillus protease (E.G.3.4.24.28) or an alkaline serine protease (E.G.3.4.21.x) or a keratinase (E.G.3.4.X.X). In one embodiment, the protease is a subtilisin. Suitable proteases include those of animal, plant or microbial origin.

Chemically modified or protein engineered mutants are also suitable. The protease may be a serine protease or a metalloprotease. For example, an alkaline microbial protease or a trypsin-like protease. In one embodiment, the present invention provides a composition comprising any one of the engineered phytase polypeptides disclosed herein or its fragment and one or more proteases.

Examples of alkaline proteases are subtilisins, especially those derived from Bacillus species, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, e.g., U.S. Pat. No. 6,287,841), subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279). Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583). Examples of useful proteases also include, but are not limited to, variants described in WO 92/19729 and WO 98/20115.

In one embodiment, the protease is selected from the group consisting of subtilisin, bacillus protease, alkaline serine protease, keratinase and Nocardiopsis protease.

It is understood that one protease unit (PU) is the amount of enzyme that releases one microgram of phenolic compounds (expressed as tyrosine equivalents) from substrate (0.6% casein solution) in one minute at pH 7.5 (40 mM _{Na2PO4 /} lactate buffer) and 40°C. This may be referred to as an assay for determining 1 PU.

In one embodiment, the present disclosure relates to a composition comprising any engineered phytase polypeptide disclosed herein or a fragment thereof and a protease. In another embodiment, the present disclosure relates to a composition comprising any engineered phytase polypeptide disclosed herein or a fragment thereof and a xylanase and a protease. In still another embodiment, the present disclosure relates to a composition comprising any engineered phytase polypeptide disclosed herein or a fragment thereof and an amylase and a protease. In yet another embodiment, the present disclosure relates to a composition comprising any engineered phytase polypeptide disclosed herein or a fragment thereof and a xylanase, an amylase and a protease.

In one embodiment, the composition comprises about 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 protease units/g composition.

In one embodiment, the composition comprises about 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-70 -15000, 15000-16000, 16000-17000, 17000-18000, 18000-20000 and more than 15000 protease units/g of composition.

The at least one direct fed microbial (DFM) may comprise at least one viable microorganism, such as a viable bacterial strain or a viable yeast or a viable fungus. Preferably, the DFM comprises at least one viable bacterium.

It is possible that the DFM may be a spore-forming bacterial strain, and thus the term DFM may consist of or contain spores, such as bacterial spores. Thus, as used herein, the term "viable microorganism" may include microbial spores, such as endospores or conidia. Alternatively, the DFM in the feed additive composition described herein may not consist of or contain microbial spores, such as endospores or conidia.

The microorganism may be a naturally occurring microorganism, or it may also be a transformed microorganism.

The DFM as described herein may comprise a microorganism from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera, and combinations thereof.

Preferably, the DFM comprises one or more bacterial strains selected from the following Bacillus species: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilis and Bacillus amyloliquefaciens.

As used herein, "Bacillus" includes all species within the "Bacillus" genus as known to those of skill in the art, including, but not limited to, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, B. brevis, and B. thuringiensis. It will be recognized that the genus Bacillus continues to undergo taxonomic recombination. Thus, the genus is intended to include species that have been reclassified, including, but not limited to, organisms such as Bacillus stearothermophilus (now named "Geobacillus stearothermophilus") or Bacillus polymyxa (now named "Paenibacillus polymyxa"). The production of resistant endospores under stressful environmental conditions is considered to be a defining characteristic of the genus Bacillus, although this property also applies to the more recently named genera Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

In another aspect, the DFM may be further combined with the following Lactococcus species: Lactococcus cremoris and Lactococcus lactis and combinations thereof.

The DFM may further be combined with the following Lactobacillus species: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei, Lactobacillus reuteri, Lactobacillus fermentum ... reuteri, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus farciminis, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, and Lactobacillus jensenii, and any combination thereof.

In another aspect, the DFM may be further combined with the following Bifidobacterium species: Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis and Bifidobacterium angulatum, and any combination thereof.

Bacteria of the following species may be mentioned: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus brevis, Enterococci, Enterococcus species and Pediococcus species, Lactobacillus species, Bifidobacterium species, Lactobacillus acidophilus, Pediococcus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Bacillus subtilis, Propionibacterium thoenii, Lactobacillus farinaceus, Lactobacillus rhamnosus, Megasphaera elsdenii, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillus salivarius salivarius. salivarius ssp. Salivarius), Propionibacterium species, and combinations thereof.

The direct fed microbial comprising one or more bacterial strains described herein may be of the same type (genus, species and strain), or may comprise a mixture of genera, species and/or strains.

(以前称为

)；枯草芽孢杆菌菌株C3102(来自

)；枯草芽孢杆菌菌株PB6(来自

)；短小芽孢杆菌(8G-134)；肠球菌NCIMB 10415(SF68)(来自

)；枯草芽孢杆菌菌株C3102(来自

和

)；地衣芽孢杆菌(来自

)；肠球菌和片球菌(来自Poultry

)；乳杆菌、双歧杆菌和/或肠球菌(来自

)；枯草芽孢杆菌菌株QST 713(来自

)；解淀粉芽孢杆菌CECT-5940(来自

和

Plus)；屎肠球菌(Enterococcusfaecium)SF68(来自

)；枯草芽孢杆菌和地衣芽孢杆菌(来自

)；乳酸菌7屎肠球菌(来自

)；芽孢杆菌菌株(来自

)；酿酒酵母(Saccharomycescerevisiae)(来自Yea-

)；肠球菌(来自Biomin

)；乳酸片球菌、肠球菌、动物双歧杆菌动物亚种、罗伊氏乳杆菌、唾液乳杆菌唾液亚种(来自Biomin

)；香肠乳杆菌(来自

)；肠球菌(来自Oralin

)；肠球菌(2个菌株)、乳酸乳球菌DSM1103(来自Probios-pioneer

)；鼠李糖乳杆菌和香肠乳杆菌(来自

)；枯草芽孢杆菌(来自

)；肠球菌(来自

)；酿酒酵母(来自LevucellSB

)；酿酒酵母(来自Levucell SC 0&

ME)；乳酸片球菌(来自Bactocell)；酿酒酵母(来自

(以前为

))；酿酒酵母NCYC Sc47(来自

SC47)；丁酸梭菌(来自Miya-

)；肠球菌(来自Fecinor和Fecinor

)；酿酒酵母NCYC R-625(来自

)；酿酒酵母(来自

)；肠球菌和鼠李糖乳杆菌(来自

)；枯草芽孢杆菌和米曲霉(来自PepSoyGen-

)；蜡样芽孢杆菌(来自

)；蜡样芽孢杆菌东洋变体(Bacillus cereus var.toyoi)NCIMB40112/CNCMI-1012(来自

)，或其他DFM，诸如地衣芽孢杆菌和枯草芽孢杆菌(来自

YC)和枯草芽孢杆菌(来自

)。Alternatively, the DFM may be combined with one or more of the products disclosed in WO 2012110778 or the microorganisms contained in these products and summarized as follows: Bacillus subtilis strain 2084 Accession No. NRRL B-50013, Bacillus subtilis strain LSSAO1 Accession No. NRRL B-50104, and Bacillus subtilis strain 15A-P4 ATCC Accession No. PTA-6507 (from Enviva

(formerly known as

); Bacillus subtilis strain C3102 (from

); Bacillus subtilis strain PB6 (from

); Bacillus pumilus (8G-134); Enterococcus NCIMB 10415 (SF68) (from

); Bacillus subtilis strain C3102 (from

and

); Bacillus licheniformis (from

); Enterococci and Pediococci (from Poultry

); Lactobacillus, Bifidobacterium and/or Enterococcus (from

); Bacillus subtilis strain QST 713 (from

); Bacillus amyloliquefaciens CECT-5940 (from

and

Plus); Enterococcus faecium SF68 (from

); Bacillus subtilis and Bacillus licheniformis (from

); Lactobacillus 7 Enterococcus faecium (from

); Bacillus strains (from

); Saccharomyces cerevisiae (from Yea-

); Enterococci (from Biomin

); Pediococcus acidilactici, Enterococcus, Bifidobacterium animalis subsp. animalis, Lactobacillus reuteri, Lactobacillus salivarius subsp. salivarius (from Biomin

); Lactobacillus fumigans (from

); Enterococci (from Oralin

); Enterococci (2 strains), Lactococcus lactis DSM1103 (from Probios-pioneer

); Lactobacillus rhamnosus and Lactobacillus farnesii (from

); Bacillus subtilis (from

); Enterococci (from

); Saccharomyces cerevisiae (from LevucellSB

); Saccharomyces cerevisiae (from Levucell SC 0&

ME); Pediococcus acidilactici (from Bactocell); Saccharomyces cerevisiae (from

(Previously

)); Saccharomyces cerevisiae NCYC Sc47 (from

SC47); Clostridium butyricum (from Miya-

); Enterococci (from Fecinor and Fecinor

); Saccharomyces cerevisiae NCYC R-625 (from

); Saccharomyces cerevisiae (from

); Enterococci and Lactobacillus rhamnosus (from

); Bacillus subtilis and Aspergillus oryzae (from PepSoyGen-

); Bacillus cereus (from

); Bacillus cereus var. toyoi NCIMB40112/CNCMI-1012 (from

), or other DFMs such as Bacillus licheniformis and Bacillus subtilis (from

YC) and Bacillus subtilis (from

).

PRO组合，后者可从丹尼斯科公司商购获得。Enviva

是芽孢杆菌菌株2084登录号NRRL B-50013、芽孢杆菌菌株LSSAO1登录号NRRL B-50104和芽孢杆菌菌株15A-P4 ATCC登录号PTA-6507的组合(如在US 7,754,469 B中所传授的-通过援引并入本文)。DFM can be

PRO combination, the latter of which is commercially available from Danisco Corporation. Enviva

It is a combination of Bacillus strain 2084 Accession No. NRRL B-50013, Bacillus strain LSSAO1 Accession No. NRRL B-50104, and Bacillus strain 15A-P4 ATCC Accession No. PTA-6507 (as taught in US 7,754,469 B - incorporated herein by reference).

The DFMs described herein may also be combined with yeast from the genus: Saccharomyces species.

Preferably, the DFM described herein comprises a microorganism that is generally recognized as safe (GRAS) and preferably GRAS approved.

One of ordinary skill in the art will readily recognize specific microbial species and/or strains from within the genera described herein that are used in the food and/or agricultural industries and that are generally considered suitable for animal consumption.

In some embodiments, it is important that the DFM is thermotolerant, i.e. heat resistant. This is particularly true when the feed is pelleted. Thus, in another embodiment, the DFM may be a thermotolerant microorganism, such as a thermotolerant bacterium, including, for example, Bacillus species.

In other aspects, it may be desirable for the DFM to comprise spore-forming bacteria, such as Bacillus, e.g., Bacillus species. Bacillus are able to form stable endospores when growth conditions are unfavorable and are highly resistant to heat, pH, moisture and disinfectants.

The DFMs described herein can reduce or prevent the intestinal establishment of pathogenic microorganisms such as Clostridium perfringens and/or Escherichia coli and/or Salmonella species and/or Campylobacter species. In other words, the DFM can be anti-pathogenic. As used herein, the term "anti-pathogenic" means that the DFM resists the effect (negative effect) of pathogens.

As described above, the DFM may be any suitable DFM. For example, the following assay "DFM Assay" may be used to determine the suitability of a microorganism as a DFM. The DFM Assay as used herein is explained in more detail in US 2009/0280090. For the avoidance of doubt, a DFM selected as an inhibitory strain (or anti-pathogenic DFM) according to the "DFM Assay" taught herein is a suitable DFM for use according to the present disclosure, i.e. a DFM for use in a feed additive composition according to the present disclosure.

Each tube is inoculated with a representative pathogen (eg, bacteria) from a representative cluster.

Supernatants from potential DFMs grown aerobically or anaerobically were added to inoculated tubes (except for controls where no supernatant was added) and incubated. After incubation, the optical density (OD) of each pathogen in the control tubes and supernatant-treated tubes was measured.

Colonies of the (potential DFM) strain producing a lower OD compared to the control (not containing any supernatant) can then be classified as suppressive strains (or anti-pathogenic DFM). Thus, the DFM assay as used herein is explained in more detail in US 2009/0280090.

Preferably, the representative pathogen used in this DFM assay may be one (or more) of the following: Clostridium, such as Clostridium perfringens and/or Clostridium difficile, and/or Escherichia coli and/or Salmonella species and/or Campylobacter species. In a preferred embodiment, the assay is performed with one or more of the following: Clostridium perfringens and/or Clostridium difficile and/or Escherichia coli, preferably Clostridium perfringens and/or Clostridium difficile. More preferably Clostridium perfringens.

Anti-pathogenic DFMs include one or more of the following bacteria and are described in WO 2013029013:

Bacillus subtilis strain 3BP5 accession number NRRL B-50510,

Bacillus amyloliquefaciens strain 918 ATCC accession number NRRL B-50508 and

Bacillus amyloliquefaciens strain 1013 ATCC accession number NRRL B-50509.

The DFM may be prepared as a culture and carrier (if used) and may be added to a ribbon or paddle mixer and mixed for about 15 minutes, although the time may be increased or decreased. The components are blended so that a uniform mixture of the culture and carrier is obtained. The final product is preferably a dry, flowable powder. The DFM comprising one or more bacterial strains may then be added to the animal feed or feed premix, added to the animal's water, or administered in other ways known in the art (preferably simultaneously with the enzymes described herein).

The proportion of individual strains included in the DFM mixture may vary from 1% to 99% and preferably from 25% to 75%.

Suitable dosages of DFM in animal feed may range from about ^1x103 to about ^1x1010 CFU/g feed, suitably between about 1x104 to about ^1x108 CFU ^/ g feed, suitably between about ^7.5x104 to about ^1x107 CFU/g feed.

In another aspect, the dose of DFM in the feed exceeds about 1 x ¹⁰³ CFU/g feed, suitably exceeds about 1 x ¹⁰⁴ CFU/g feed, suitably exceeds about 5 x ¹⁰⁴ CFU/g feed, or suitably exceeds about 1 x ¹⁰⁵ CFU/g feed.

The dosage of DFM in the feed additive composition is from about 1x10 ³ CFU/g composition to about 1x10 ¹³ CFU/g composition, preferably 1x10 ⁵ CFU/g composition to about 1x10 ¹³ CFU/g composition, more preferably between about 1x10 ⁶ CFU/g composition to about 1x10 ¹² CFU/g composition, and most preferably between about 3.75x10 ⁷ CFU/g composition to about 1x10 ¹¹ CFU/g composition. In another aspect, the dosage of DFM in the feed additive composition is greater than about 1x10 ⁵ CFU/g composition, preferably greater than about 1x10 ⁶ CFU/g composition and most preferably greater than about 3.75x10 ⁷ CFU/g composition. In one embodiment, the dosage of DFM in the feed additive composition is greater than about 2x10 ⁵ CFU/g composition, suitably greater than about 2x10 ⁶ CFU/g composition, suitably greater than about 3.75x10 ⁷ CFU/g composition.

In still another aspect, a granular feed additive composition is disclosed for use in an animal feed, the animal feed comprising at least one polypeptide having phytase activity as described herein, alone or in combination with at least one direct-fed microorganism, or in combination with at least one other enzyme, or in combination with at least one direct-fed microorganism and at least one other enzyme, wherein the feed additive composition comprises may be in any form, such as granular particles. Such granular particles may be produced by a process selected from the group consisting of high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spray coating, spray drying, freeze drying, granulation, spray cooling, vortex disk atomization, agglomeration, tableting, or any combination of the foregoing methods.

Furthermore, the particles of the granular feed additive composition may have an average diameter greater than 50 microns and less than 2000 microns.

Those skilled in the art will appreciate that animal feeds may include plant materials for poultry, swine, ruminants, aquaculture and pets, such as corn, wheat, sorghum, soybeans, canola, sunflower, or a mixture of any of these plant materials or plant protein sources. It is expected that animal performance parameters such as growth, feed intake and feed efficiency, as well as improved uniformity, reduced ammonia concentrations in animal sheds and thus improved animal welfare and health will all be improved.

Thus, a method for improving the nutritional value of animal feed is disclosed, wherein any of the engineered phytases or fragments thereof as described herein may be added to the animal feed.

The phrase "effective amount" as used herein refers to the amount of an active agent (such as a phytase, e.g. any of the engineered phytase polypeptides disclosed herein) required to confer improved performance on one or more parameters to an animal, either alone or in combination with one or more other active agents (such as but not limited to one or more additional enzymes, one or more DFMs, one or more essential oils, etc.).

As used herein, the term "animal performance" can be determined by any indicator such as, but not limited to, feed efficiency and/or weight gain of the animal, and/or by feed conversion ratio and/or by digestibility of nutrients in the feed (e.g., amino acid digestibility or phosphorus digestibility) and/or digestible or metabolizable energy in the feed, and/or by nitrogen retention and/or by the ability of the animal to avoid the negative effects of disease or by the immune response of the subject.

Animal performance characteristics may include, but are not limited to: body weight; weight gain; mass; body fat percentage; height; body fat distribution; growth; growth rate; egg size; egg weight; egg quality; egg production rate; mineral absorption; mineral excretion, mineral retention; bone density; bone strength; feed conversion ratio (FCR); average daily feed intake (ADFI); average daily gain (ADG); retention and/or secretion of any one or more of copper, sodium, phosphorus, nitrogen and calcium; amino acid retention or absorption; mineralization, bone mineralization carcass yield and carcass quality.

"Improved animal performance in one or more parameters" means increased feed efficiency and/or increased weight gain and/or decreased feed conversion ratio and/or improved digestibility of nutrients or energy in the feed and/or improved nitrogen retention and/or improved ability to avoid the negative effects of necrotic enteritis and/or improved immune response in the subject due to the use of a feed comprising the feed additive composition as described herein, compared to a feed not comprising the feed additive composition described herein.

Preferably, by "improved animal performance" is meant an increase in feed efficiency and/or an increase in weight gain and/or a decrease in feed conversion ratio. As used herein, the term "feed efficiency" refers to the amount of weight gain an animal experiences when the animal is fed ad libitum or a specified amount of food over a period of time. As used herein, "improvement in an indicator" or "improved indicator" refers to an improvement in at least one parameter listed under an indicator defined for an animal.

By "increased feed efficiency" is meant that the use of the feed additive composition according to the invention in feed results in increased weight gain per unit of feed intake compared to animals fed in the absence of the feed additive composition.

As used herein, the term "feed conversion ratio" refers to the amount of feed fed to an animal to increase the animal's body weight by a specified amount.

Improved feed conversion means lower feed conversion ratio.

"Lower feed conversion ratio" or "improved feed conversion ratio" means that the use of the feed additive composition in the feed results in a lower amount of feed required to be fed to the animal to increase the animal's weight by a specified amount compared to the amount of feed required to increase the animal's weight by the same amount when the feed did not contain the feed additive composition.

The improvement in performance parameters may be relative to a control wherein the feed used does not contain phytase.

The term tibia ash refers to a quantitative method for bone mineralization. This parameter gives an indication of whether phosphorus is deficient (e.g., levels should be low in a phosphorus-deficient negative control diet) or sufficient (e.g., levels in a phytase treatment comparable to a positive control diet that meets the phosphorus requirements of broilers).

The term "phosphorus-deficient diet" refers to a diet in which the phosphorus level is insufficient to meet the nutritional requirements of the animal, for example, a diet formulated with phosphorus levels far below those recommended by the National Research Council (NRC) or broiler producers. The mineral levels in the animal's feed are below those required for optimal growth. If the diet lacks phosphorus, calcium is also not absorbed by the animal. Excessive Ca can lead to poor phosphorus (P) digestibility and the formation of insoluble mineral-phytate complexes. Deficiencies in both P and Ca can lead to reduced bone integrity, subnormal growth, and ultimately weight loss.

The term "mineralization" or "mineralization" encompasses the deposition of minerals or the release of minerals. Minerals may be deposited or released from an animal. Minerals may be released from feed. Minerals may include any mineral essential to an animal's diet, and may include calcium, copper, sodium, phosphorus, iron, and nitrogen. In a preferred embodiment, the use of the engineered phytase polypeptide of the present invention or a fragment thereof in food or feed results in an increased deposition of calcium in an animal, particularly in bone.

As used herein, nutrient digestibility means the fraction of nutrients disappearing from the gastrointestinal tract or a specific segment of the gastrointestinal tract (e.g., the small intestine). Nutrient digestibility can be measured as the difference between the nutrients administered to the subject and the nutrients discharged in the feces of the subject, or the difference between the nutrients administered to the subject and the nutrients retained in the digesta in a specified segment of the gastrointestinal tract (e.g., the ileum).

As used herein, nutrient digestibility can be measured as follows: by the difference between the amount of nutrient intake over a period of time and the amount of nutrient excreted as completely collected by excrement; or using an inert marker that is not absorbed by the animal and allows researchers to calculate the amount of nutrients that disappear in the entire gastrointestinal tract or a segment of the gastrointestinal tract. Such inert markers can be titanium dioxide, chromium oxide, or acid-insoluble ash. Digestibility can be expressed as a percentage of the nutrient in the feed, or as a mass unit of digestible nutrient/mass unit of nutrient in the feed.

As used herein, nutrient digestibility encompasses phosphorus digestibility, starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility. Digestible phosphorus (P) can be defined as ileal digestible P, which is the proportion of total P intake that is absorbed by the animal in the terminal ileum, or fecal digestible P, which is the proportion of total P intake that is not excreted in the feces.

As used herein, the term "survival rate" means the number of subjects that survive. The term "improved survival rate" is another way of saying "reduced mortality rate."

As used herein, the term "carcass yield" means the amount of carcass measured as a proportion of live weight after a commercial or experimental slaughter process. The term carcass means the body of an animal that has been slaughtered for consumption and from which the head, viscera, limb parts, and feathers or skin have been removed. As used herein, the term meat yield means the amount of edible meat measured as a proportion of live weight, or the amount of a specified cut of meat measured as a proportion of live weight.

The terms "carcass quality" and "meat quality" are used interchangeably and refer to compositional quality (fat to lean ratio) as well as palatability factors such as visual appearance, smell, firmness, juiciness, tenderness and flavor. For example, producing poultry without "woody breast". In the United States, muscle abnormalities in a small number of chickens lead to the quality problem of woody breast, a condition that causes the chicken breast to be difficult to touch and often pale in color and of poor texture. Woody breast does not pose any health or food safety risks to people, and the health of the chicken itself is not negatively affected.

"Increased weight gain" refers to an increase in body weight of an animal fed a feed comprising the feed additive composition compared to an animal fed a feed not containing the feed additive composition.

In the context of the present invention, the term "pet food" is intended to be understood as meaning food for domestic animals such as, but not limited to, dogs, cats, gerbils, hamsters, chinchillas, fancy rats, guinea pigs; avian pets such as canaries, parakeets and parrots; reptile pets, such as turtles, lizards and snakes; and aquatic pets, such as tropical fish and frogs.

The terms "animal feed composition", "feed", "feedstock" and "fodder" are used interchangeably and may comprise one or more feed materials selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains, such as maize or sorghum; b) by-products from cereals, such as corn gluten meal, distillers dried grains with solubles (DDGS), particularly corn-based distillers dried grains with solubles (cDDGS), wheat bran, semolina, wheat short meal, rice bran, rice hulls, oat hulls, palm kernels and citrus pulp; c) proteins obtained from sources such as soy, sunflower, peanut, lupin, pea, bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from plant and animal sources; and/or e) minerals and vitamins.

The engineered phytase polypeptides or fragments thereof as described herein or feed additive compositions comprising such engineered phytase polypeptides or fragments thereof can be used as feed or in the preparation of feed.

Thus, described are dry enzyme compositions for use in animal feed comprising any of the engineered phytase polypeptides or fragments thereof described herein.

Also described are liquid enzyme compositions for use in animal feed, comprising any of the engineered phytase polypeptides or fragments thereof as described herein.

The terms "feed additive composition" and "enzyme composition" are used interchangeably herein.

Depending on the use and/or the mode of application and/or the mode of administration, the feed may be in the form of a solution or in solid form or in semisolid form.

In a preferred embodiment, the enzyme or feed additive composition described herein is mixed with a feed component to form a feed.

As used herein, "feed component" means all or part of a feed. A part of a feed may mean one ingredient of the feed or more than one (eg 2 or 3 or 4 or more) ingredients of a feed.

In one embodiment, the term "feed component" encompasses a premix or a premix ingredient. Preferably, the feed may be a fodder or a premix thereof, a compound feed or a premix thereof. The feed additive composition may be mixed with a compound feed, a compound feed component or into a premix of a compound feed or into a fodder, a fodder component or a premix of a fodder.

Forage covers plants that have been cut. In addition, forage includes silage, compressed and pelleted feeds, oils and mixed rations, and also includes sprouted grains and legumes.

Suitably, a premix as referred to herein may be a composition consisting of minor ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients. A premix is typically a composition suitable for blending into a commercial ration.

As used herein, the term "contacting" refers to applying any of the engineered phytase polypeptides or fragments thereof (or a composition comprising any of the engineered phytase polypeptides or fragments thereof) indirectly or directly to a product (e.g., feed). Examples of usable application methods include, but are not limited to, treating the product in a material comprising a feed additive composition, applying directly by mixing the feed additive composition with the product, spraying the feed additive composition onto the surface of the product, or immersing the product in a formulation of the feed additive composition. In one embodiment, the feed additive composition of the present invention is preferably mixed with a product (e.g., a feed). Alternatively, the feed additive composition may be included in an emulsion or original ingredient of the feed. For some applications, it is important that the composition is available on or available for the surface of the product to be affected/treated. This allows the composition to impart performance benefits.

In some aspects, any engineered phytase polypeptide or fragment thereof can be used for pretreatment of food or feed. For example, a feed having 10%-300% moisture is mixed and incubated with the engineered phytase polypeptide or fragment thereof at 5°C-80°C (preferably 25°C-50°C, more preferably between 30°C-45°C), wherein the incubation is 1 minute to 72 hours under aerobic conditions or 1 day to 2 months under anaerobic conditions. The pretreated material can be fed directly to the animal (so-called liquid feeding). The pretreated material can also be steam pressed into pellets at elevated temperatures (60°C-120°C). The engineered phytase polypeptide or fragment thereof can be impregnated into feed or food materials by a vacuum coater.

Any of the engineered phytase polypeptides or fragments thereof described herein (or compositions comprising such engineered phytase polypeptides or fragments thereof) and a controlled amount of the enzyme may be used to spread, coat and/or impregnate a product (eg, a feed or an original ingredient of a feed).

On the other hand, the feed additive composition can be homogenized to produce a powder. The powder can be mixed with other components known in the art. The powder or a mixture comprising the powder can be forced through a die, and the resulting strands are cut into suitable pellets of varying lengths.

Optionally, the granulation step may include a steam treatment or conditioning stage before forming the pellets. The mixture comprising the powder may be placed in a conditioner, for example, a mixer with steam injection. The mixture is heated in the conditioner to a specified temperature, such as 60°C-100°C, a typical temperature would be 70°C, 80°C, 85°C, 90°C or 95°C. The residence time may vary from a few seconds to a few minutes. It should be understood that any of the engineered phytase polypeptides or fragments thereof described herein (or a composition comprising any of the engineered phytase polypeptides or fragments thereof) is suitable for addition to any appropriate feed material.

In other embodiments, the particles may be introduced into a feed pelleting process, wherein the feed pre-treatment process may be carried out at between 70°C and 95°C, such as between 85°C and 95°C for up to several minutes.

In some embodiments, any of the engineered phytase polypeptides or fragments thereof may be present in the feed in the range of 1 ppb (parts per billion) to 10% (w/w) based on pure enzyme protein. In some embodiments, the engineered phytase polypeptide or fragment thereof is present in the feed in the range of 1-100 ppm (parts per million). A preferred dosage may be 1-20 g of the engineered phytase polypeptide or fragment thereof per ton of feed product or feed composition, or a final dosage of 1-20 ppm of the engineered phytase polypeptide or fragment thereof in the final feed product.

Preferably, the engineered phytase polypeptide or fragment thereof should be present in the feed at least about 50-10,000 FTU/kg, equivalent to about 0.1 to 20 mg of engineered phytase polypeptide or fragment thereof protein/kg.

Ranges can include, but are not limited to, any combination of the lower and upper limits discussed above.

The preparation and/or formulation comprising any one of the engineered phytase polypeptides or fragments thereof and compositions described herein can be prepared in any suitable manner to ensure that the preparation comprises active phytase. Such preparations can be liquids, dry powders or granules, which can be uncoated/unprotected, or can involve the use of a thermal protective coating, depending on the processing conditions. As described above, engineered phytase polypeptides and fragments thereof can be formulated inexpensively on solid carriers without the particular need for a protective coating, and remain active throughout the conditioning and granulation process. When certain areas where harsh conditions are used require or if conditions permit (e.g., in the case of ultra-conditioned feeds above 90°C), a protective coating providing additional thermal stability when applied in solid form may be beneficial for obtaining granulation stability.

The feed additive composition described herein may be formulated as a dry powder or granules as described in WO 2007/044968 (referred to as TPT granules) or in WO 1997/016076 or WO 1992/012645 (each of which is incorporated herein by reference).

In one embodiment, the feed additive composition can be formulated as a granule for a feed composition, the granule comprising: a core; an active agent (e.g., a phytase, such as any of the engineered phytase polypeptides disclosed herein); and at least one coating, wherein the active agent of the granule remains at least 50% active, at least 60% active, at least 70% active, at least 80% active after being subjected to one or more conditions selected from the following: a) a feed pelleting process, b) a steam heating feed pretreatment process, c) storage, d) storage as an ingredient in an unpelleted mixture, and e) storage as an ingredient in a feed base mixture or feed premix comprising at least one compound selected from the following: trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds that produce an acidic or alkaline feed base mixture or feed premix.

With respect to the granules, at least one coating may comprise a moisture hydrated material that accounts for at least 55% w/w of the granule; and/or at least one coating may comprise two coatings. The two coatings may be a moisture hydrated coating and a moisture barrier coating. In some embodiments, the moisture hydrated coating may account for 25% w/w to 60% w/w of the granule, and the moisture barrier coating may account for 2% w/w to 15% w/w of the granule. The moisture hydrated coating may be selected from an inorganic salt, sucrose, starch, and maltodextrin, and the moisture barrier coating may be selected from a polymer, a gum, whey, and starch.

In other embodiments, the particles may be introduced into a feed pelleting process, wherein the feed pre-treatment process may be carried out at between 70°C and 95°C, such as between 85°C and 95°C for up to several minutes.

The feed additive composition can be formulated as a granule for animal feed, the granule comprising: a core; an active agent, the active agent of the granule remaining at least 80% active after storage and after a steam-heated pelleting process of which the granule is an ingredient; a moisture barrier coating; and a moisture-hydrated coating comprising at least 25% w/w of the granule, the granule having a water activity of less than 0.5 prior to the steam-heated pelleting process.

The granules may have a moisture barrier coating selected from polymers and gums and the moisture hydrating material may be an inorganic salt. The moisture hydrating coating may comprise 25% to 45% w/w of the granules and the moisture barrier coating may comprise 2% to 10% w/w of the granules.

Alternatively, the composition is in a liquid formulation suitable for consumption, preferably such liquid consumable contains one or more of: a buffer, salts, sorbitol and/or glycerol.

Furthermore, the feed additive composition may be formulated by applying (eg, spraying) the enzyme onto a carrier substrate such as ground wheat.

In one embodiment, the feed additive composition may be formulated as a premix. By way of example only, a premix may include one or more feed components, such as one or more minerals and/or one or more vitamins.

In one embodiment, the direct fed microbial ("DFM") and/or engineered phytase polypeptide or fragment thereof is formulated with at least one physiologically acceptable carrier selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, _Na2SO4 , _talc , PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate, and mixtures thereof.

The carrier that can be used to make animal feed pellets containing any engineered phytase polypeptide or fragment disclosed herein is preferably selected from feed liquid or solid carriers that allow transformation into a defined shape similar to conventional feed pellets. In some non-limiting embodiments, the following two types of liquid or solid carriers can be used: (i) carriers used in solvent or dispersant media; and (ii) carriers that can be melted.

The first type of carrier used in the solvent or dispersion medium includes: hydrocolloids, preferably water-soluble derivatives of cellulose, more preferably carboxymethylcellulose, hydroxypropylcellulose, hydroxyethylcellulose and hydroxymethylcellulose; natural or synthetic polysaccharides, preferably gum arabic, tragacanth, carrageenan, dextrin, starch, xanthan gum and alginates; sugars; molasses and brewer's grains; lignin sulfonates; cereal or seaweed flour; crystallizable inorganic compounds, preferably lime, gypsum, sodium silicate, calcium carbonate and silicon dioxide; gelatin; tanned protein; polyvalent cationic salts of natural or synthetic polyacids; and drying oils and mastic obtained by combining drying oils and fillers.

Some carriers are used with a crosslinking agent. Preferred crosslinking agents include aldehydes for proteins, and salts or oxides of divalent or trivalent metals for alginates, xanthan gum, molasses, brewer's grains, and other hardeners or curing agents known to those skilled in the art to be suitable for use in adhesives.

The second type of carrier that can be melted includes: fatty acids and alcohols; hydrogenated vegetable and animal fats; glycerides; paraffins; natural and synthetic waxes; and synthetic polymers, preferably polyethylene glycols and polyvinyl acetates. Of all the binders, the most preferred are molasses, brewer's grains, fatty acids, hydrogenated vegetable or animal fats, gypsum and paraffin.

The following are non-limiting examples of therapeutic or nutritional agents that can be incorporated into the mixture to be formed into pellets that can be used to make animal feed pellets containing any of the engineered phytase polypeptides or fragments disclosed herein: mineral additives such as phosphorus, sulfur, magnesium, zinc, copper, cobalt, sodium, potassium, chlorine, iron, calcium, iodine, molybdenum, selenium, nickel and vanadium; vitamins such as vitamins A, B, D and E; energy-producing foods such as glucose, long-chain fatty acids and volatile fatty acids; yeast; growth factors; enzymes (such as any enzyme disclosed herein); DFMs (such as any DFMs disclosed herein); peptides, such as, in particular, growth hormones; and food adjuvants such as sodium bicarbonate, sorbitol, propylene glycol, betaine and sodium propionate.

Other non-limiting examples of therapeutic or nutritional agents that can be used to make animal feed pellets containing any of the engineered phytase polypeptides or fragments disclosed herein and that can be incorporated into the mixture to be formed into pellets include: essential amino acids, their salts, their derivatives and analogs thereof, preferably methionine and lysine; vitamins; and pharmaceutically active ingredients or components, such as antibiotics.

In some embodiments, methionine is used as a therapeutic agent or nutrient for making animal feed pellets containing any engineered phytase polypeptide or fragment disclosed herein and can be incorporated into a mixture to be formed into pellets. Methionine can be a methionine supplement formulation comprising at least one additional feed raw material, such as L-methionine. Another example is where the methionine is DLM (i.e., DL-methionine) or HMTBA (i.e., 2-hydroxy-4-(methylthio)butyric acid). Methionine can be in the form of L-methionine, such as in the form of a synthetic methionine source. It can be all of its salt forms of L-methionine, its analogs (e.g., 2-hydroxy-4-methylthiobutyric acid or all of its salt forms), derivatives (e.g., 2-hydroxy-4-methylthiobutyric acid isopropyl ester or any other ester) or mixtures thereof. The dosage and days of feeding the methionine are given above, and are also applicable to improving meat quality for all embodiments. In particular, methionine is used in a dosage of 0.1-1 g per kg body weight per day before slaughter, such as 0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or even 1 g total methionine. On the other hand, methionine is used in a dosage of 0.3%-0.6% higher than the recommended daily dose.

It should be noted that any of the engineered phytase polypeptides and fragments thereof may be used in grain applications, such as in grain processing for non-food/feed applications such as ethanol production.

Non-limiting examples of the compositions and methods disclosed herein include:

1. An engineered phytase polypeptide or a fragment thereof comprising phytase activity, wherein the phytase polypeptide or the fragment thereof has at least 82% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

2. The engineered phytase polypeptide or fragment thereof of Example 1, wherein the amino acid sequence of the engineered phytase polypeptide or fragment thereof has a Hidden Markov Model (HMM) score of at least about 1200, as shown in Table 11 for high Tm phytase clade polypeptides or fragments thereof.

3. An engineered phytase polypeptide or a core domain fragment thereof, wherein the engineered phytase polypeptide or a core domain fragment thereof has at least 78% sequence identity with amino acid positions 14-325 of the amino acid sequence shown in SEQ ID NO: 1.

4. An engineered phytase polypeptide or fragment thereof (e.g., those described in Examples 1, 2 or 3) having a feed pelleting recovery of at least about 50% when applied in MLA at 95°C for 30 seconds using the standard feed pelleting recovery test as described in Example 5.

5. The engineered phytase polypeptide or fragment thereof of embodiment 1 or 2, wherein the phytase polypeptide or fragment thereof has a feed pelleting recovery of at least about 50% when applied in MLA at 95°C for 30 seconds using a standard feed pelleting recovery test as described in Example 5.

6. An engineered phytase polypeptide or fragment thereof (e.g., those described in Examples 1, 2, 3, 4 or 5), having a ratio of feed pelleting recovery of at least about 0.7 when applied in MLA at 95°C for 30 seconds compared to at 80°C for 30 seconds using the standard feed pelleting test as described in Example 5.

7. The engineered phytase polypeptide or fragment thereof of embodiment 1, 2, 3, 4, 5 or 6, having a ratio of feed pelleting recovery of at least about 0.7 when applied in MLA at 95°C for 30 seconds compared to at 80°C for 30 seconds using the standard feed pelleting test as described in Example 5.

8. The engineered phytase polypeptide or fragment thereof of embodiment 1, 2, 3, 4, 5, 6 or 7, wherein the polypeptide or fragment thereof comprises a Tm temperature of at least about 92.5°C under the conditions determined using differential scanning calorimetry as described in Example 3.

9. The engineered phytase polypeptide or fragment thereof of embodiment 1, 2, 3, 4, 5, 6, 7, or 8, wherein the polypeptide or fragment thereof comprises a specific activity of at least about 100 U/mg at pH 3.5 under the assay conditions described in Example 3.

10. An animal feed, feed, feed additive composition or premix comprising an engineered phytase polypeptide or fragment thereof as described in embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein the engineered phytase polypeptide or fragment thereof is used (i) alone, or (ii) in combination with a direct-fed microbial comprising at least one bacterial strain, or (iii) in combination with at least one other enzyme, or (iv) in combination with a direct-fed microbial comprising at least one bacterial strain and at least one other enzyme, or (v) any of (i), (ii), (iii) or (iv), further comprising at least one other feed additive component, and optionally the engineered phytase polypeptide or fragment thereof is present in an amount of at least about 0.1 g/ton of feed.

11. A recombinant construct comprising a regulatory sequence that functions in a production host, the regulatory sequence being operably linked to a nucleotide sequence encoding an engineered phytase polypeptide or a fragment thereof as described in Examples 1, 2, 3, 4, 5, 6, 7, 8, or 9.

12. The recombinant construct of embodiment 11, wherein the production host is selected from the group consisting of bacteria, fungi, yeast, plants and algae.

13. A method for producing an engineered phytase polypeptide or fragment thereof, the method comprising:

(a) transforming a production host with the recombinant construct as described in Example 11; and

(b) culturing the production host of step (a) under conditions such that the engineered phytase polypeptide or fragment thereof is produced.

14. The method of embodiment 13, wherein the engineered phytase polypeptide or fragment thereof is optionally recovered from the production host.

15. A culture supernatant containing phytase, wherein the culture supernatant containing phytase is obtained by the method described in Example 13 or 14.

16. A polynucleotide sequence encoding the engineered phytase polypeptide or fragment thereof as described in embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9.

17. A dry enzyme composition for use in animal feed, comprising the engineered phytase polypeptide or fragment thereof of embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9.

18. The dry enzyme composition of embodiment 17, wherein the dry enzyme composition is a granulated feed additive composition.

19. A liquid enzyme composition for use in animal feed, comprising the engineered phytase polypeptide or fragment thereof of embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9.

20. A method for improving the nutritional value of animal feed, wherein the engineered phytase or fragment thereof as described in embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9 is added to the animal feed.

21. A method for improving animal performance in one or more parameters, the method comprising administering to the animal an effective amount of the engineered phytase polypeptide of embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9 or the animal feed, feedstuff, feed additive composition or premix of embodiment 10 or 11.

Examples

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D Edition, John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide a general dictionary of many of the terms used in the present disclosure to those of skill in the art.

The present disclosure is further defined in the following examples. It should be understood that these examples, although indicating certain embodiments, are given by way of illustration only. From the above discussion and examples, those skilled in the art will be able to ascertain the essential characteristics of the present disclosure and that various changes and modifications may be made to adapt to various uses and conditions without departing from the spirit and scope of the present disclosure.

Example 1

Production of phytase molecules

BP重组技术将所述基因引入pDonor221载体(英杰公司(Invitrogen)，美国)。将所得的进入质粒与目的载体pTTTpyr2重组，得到最终的表达载体。除了pyrG基因被pyr2基因代替外，pTTTpyr2类似于之前描述的pTTTpyrG载体(PCT公布WO 2011/063308)。载体pTTTpyr2含有里氏木霉cbhI启动子和终止子区域、构巢曲霉(Aspergillus nidulans)amdS选择标记、里氏木霉pyr2选择标记和来自里氏木霉的端粒序列(用于复制)。这些质粒在大肠杆菌TOP10细胞(英杰公司(Invitrogen)，美国)中增殖，并纯化DNA并进行序列验证。DNA manipulations were performed to generate phytase genes using molecular biology techniques known in the art. Polynucleotide fragments corresponding to the various phytase coding sequences were synthesized using preferred codons of the fungal expression host Trichoderma reseei (T. reesei) and randomly reassembled using PCR techniques. The signal sequence of the pep1 aspartic protease from Trichoderma reesei (SEQ ID NO: 63), artificially interrupted by the pep1 intron, was introduced into the N-terminus (5' end) of each phytase gene sequence. The phytase gene was cloned using the DNA sequence of the manufacturer according to the supplier's recommendations.

The gene was introduced into the pDonor221 vector (Invitrogen, USA) by BP recombination technology. The resulting entry plasmid was recombined with the destination vector pTTTpyr2 to obtain the final expression vector. Except that the pyrG gene was replaced by the pyr2 gene, pTTTpyr2 was similar to the previously described pTTTpyrG vector (PCT publication WO 2011/063308). The vector pTTTpyr2 contains the cbhI promoter and terminator region of Trichoderma reesei, the amdS selection marker of Aspergillus nidulans, the pyr2 selection marker of Trichoderma reesei, and the telomere sequence (for replication) from Trichoderma reesei. These plasmids were propagated in Escherichia coli TOP10 cells (Invitrogen, USA), and the DNA was purified and sequence verified.

All fungal manipulations, including high-throughput transformation, inoculation, fermentation, and harvesting, were performed in 96-well microtiter plates (MTPs). Plasmids were transformed into appropriate Trichoderma reesei host strains using the polyethylene glycol (PEG)-protoplast method. Briefly, a total volume of 50 μL of a transformation mixture containing approximately 0.5-2 μg DNA and 5×10 ⁶ protoplasts was treated with 200 μL of a 25% PEG solution and then diluted with an equal volume of a 1.2 M sorbitol/10 mM Tris/10 mM CaCl ₂ (pH 7.5) solution. The protoplasts were then regenerated in a liquid growth medium containing sorbitol to maintain osmotic pressure. 100 μl of the transformation mixture was transferred to a 96-well MTP containing 300 μl of minimal medium supplemented with sorbitol (0.30 M–0.84 M). The plates were grown in a shaker incubator at 28°C, 80% humidity for 3 days until fungal mycelium was established. If necessary, 20 μL of the grown culture was transferred to fresh minimal medium containing 10 mM acetamide to enhance the selection pressure and grown for another 2 days.

To express the phytase protein, the transformed T. reesei strain was cultured as follows: 20 μl of liquid culture was used to inoculate 400 μl of production medium (9 g/L casamino acids, 10 g/L (NH ₄ ) ₂ SO ₄ , 4.5 g/L KH ₂ PO ₄ , 1 g/L MgSO ₄ *7H ₂ O, 1 g/L CaCl ₂ *2H ₂ O, 33 g/L PIPPS buffer (pH 5.5), 0.25% T. reesei trace elements (100%: 175 g/L citric acid (anhydrous), 200 g/L FeSO ₄ *7H ₂ O, 16 g/L ZnSO ₄ *7H ₂ O, 3.2 g/L CuSO ₄ *5H ₂ O, 1.4 g/L MnSO4*H ₂ O, 0.8 g/L H ₃ BO ₃ ). The MTPs were incubated in a shaking incubator under the same growth conditions as described above. After 5 days of fermentation, the culture was filtered by centrifugation using a hydrophilic PVDF membrane to obtain a clear supernatant for analysis of recombinant phytase.

Example 2

Preparation and characterization of phytase

Protein purification and standardization

Cultivate the Trichoderma reesei strain of encoding recombinant phytase as described above, and use clarifying supernatant to purify phytase.The culture supernatant filtered is diluted 5 times with washing buffer (25mM sodium acetate, pH 5.5), and be loaded into the cation exchange resin (WorkBeads 40S from Bio-Works, Inc., U.S.) of purified water balance in MTP filter plate (Millipore (Millipore) Multiscreen Solvinert deep-well filter plate 96 holes MTP, 0.45uM hydrophilic membrane, #MDRLN0410).MTP is placed in centrifuge, effluent is abandoned during centrifugation (100x g) of 1 minute.During centrifugation (100x g) of 1 minute, elution buffer (25mM sodium acetate, 0.5M NaCl, pH 5.5) elution phytase protein sample is used. In a 96-well UV MTP (Costar, 3635), samples in the protein purification step were diluted 5-fold to a final volume of 100 μl with sodium acetate buffer (25 mM sodium acetate, 0.5 M NaCl, pH 5.5). The absorbance of the sample was measured at 280 nm, and the protein concentration was calculated according to the standard curve of the phytase protein, and the concentration range was known to be 0-1750 ppm. Based on the determined protein concentration, all samples from the purification were diluted to a target value of 150 ppm in a 96-well MTP in a buffer (100 mM sodium acetate, 0.5 M NaCl pH 5.5) and stored at 5° C. until used in the assay described below.

Determine the phytase protein concentration in each sample by reverse phase HPLC (RP-HPLC).Standardized samples are loaded on Agilent (Agilent) Zorbax300 post (SB-C3 2.1x 50mm) on Agilent 1260HPLC.According to Table 2, the gradient of solvent A (0.1v/v%TFA in water) and solvent B (0.07%TFA in acetonitrile) is applied.The sample injection volume is 10 μ l, and the column temperature is 60 ℃, and the flow velocity is 1mL/min.Measure the absorbance of eluent at 220nm, and use ChemStation software (Agilent Technologies (Agilent Technologies)) to integrate.Determine the protein concentration of phytase sample according to the standard curve of phytase protein, and the known concentration range is 0-350ppm.

软件10.2.4版本计算摩尔消光系数。Purification of samples (phytase PHY-11895, PHY-11932 and PHY-12663, and extracts of commercial products QuantumBlue 5G and Natuphos E 10000G (extraction method described in Example 5)) was performed as follows. The sample was buffer exchanged on a PD10 column (pre-equilibrated with a buffer (10-30 mM sodium acetate, pH 5.5)) and subsequently purified using hydrophobic interaction chromatography HIC. Depending on the sample, one of the following HIC columns (phenyl HP XK26 or HiTrap phenyl HP or phenyl 15, HR5/5) was used. The HIC column was pre-equilibrated in a loading buffer (20 mM sodium acetate buffer, pH 5.5, containing 1.0-1.3 M ammonium sulfate). The bound phytase protein was eluted using a linear gradient of ammonium sulfate in 10 mM sodium acetate at pH 5.5. The fractions collected from the HIC column were buffer exchanged using Sephadex G25 M, XK50/35 or PD10 columns (pre-equilibrated with buffer (10-30 mM sodium acetate, pH 5.5)). It was estimated that the final purity of all purified phytase samples (phytase PHY-11895, PHY-11932 and PHY-12663 and extracts of the commercial products Quantum Blue 5G and Natuphos E 10000G) was more than 95%. The protein concentration in the final purified sample was determined by spectrophotometric measurement of the absorbance at 280 nm and using the calculated extinction coefficient. For the two commercial products (Quantum Blue 5G and Natuphos E10000G), the calculated extinction coefficients of two closely related public phytase sequences (SEQ ID NO:61 and SEQ ID NO:60) were used. Using

The molar extinction coefficient was calculated using software version 10.2.4.

Example 3

In vitro assay of phytase

The following assays were used to measure various properties of high Tm-phytase clade polypeptides and fragments thereof, as well as commercially available phytases.

Reference phytase activity (FTU)

The activity of the phytase samples was determined by the reference phytase activity method (FTU). The following modified ISO30024 procedure was used: "Animal feeding stuffs-Determination of phytase activity": In preparation for analysis, the liquid phytase samples were diluted in assay buffer (250 mM sodium acetate, 1 mM CaCl2 and 0.01% Tween-20, pH 5.5) to obtain measurements within the linear range of the phosphate standard curve in the subsequent FTU phytase assay. For solid samples, 1.0 g of sample was weighed and extracted in 100 mL of assay buffer by mixing on a magnetic stirrer for 20 minutes. The supernatant was collected after filtration (glass fiber filter, GA-55, Advantec) and further diluted to about 0.04 FTU/mL. The analysis of the samples was performed according to the following procedure: 1 mL of the diluted phytase sample was mixed with 2 mL of 7.5 mM IP6 substrate solution (sodium phytate from rice, Shanghai AZ Import and Export, Zhejiang Orient PhyticAcid Co. Ltd #Z0201301181) in assay buffer and incubated in a water bath at 37°C for 60 min. The reaction was terminated with 2 mL of acidic molybdate/vanadate reagent and the content of inorganic phosphate was quantified spectrophotometrically at 415 nm. The results were corrected by subtracting the absorbance of the buffer blank from the absorbance of the phytase sample. A standard curve for phosphate was generated from dry potassium hydrogen phosphate and used to calculate the amount of phosphate released by each sample. One FTU is defined as the amount of phytase that produces 1 micromole of phosphate per minute.

Specific activity against IP6 substrate at pH 3.5 or 5.5

The phytase activity of phytase was determined using IP6 substrate solution (sodium phytate from rice, Shanghai AZ Import and Export, Zhejiang Orient Phytic Acid Co. Ltd #Z0201301181). For evaluation at pH 5.5, phytase samples at a concentration of 150 ppm were serially diluted to a final concentration of 0.18 ppm using 100 mM sodium acetate buffer, 0.025% Tween-20 and 0.05 mM _CaCl2 (pH 5.5) in 384 MTP prior to analysis. To each well of the 384 MTP was added 47 μL of IP6 substrate (0.20 mM) in 100 mM sodium acetate, 0.025% Tween 20 and 0.05 mM CaCl ₂ (pH 5.5) and 3 μL of the diluted phytase sample to a final volume of 50 μL.

To assess activity at pH 3.5, phytase samples at a concentration of 150 ppm were serially diluted to a final concentration of 0.11 ppm in 100 mM sodium acetate buffer, 0.025% Tween-20 and 0.05 mM _CaCl2 (pH 5.5) in 384 MTP prior to analysis. 47 μL of IP6 substrate (0.20 mM) in 100 mM glycine, 0.025% Tween-20 and 0.05 mM _CaCl2 (pH 3.3) was added to each well of the 384 MTP, and 3 μL of the diluted phytase sample was added to a final volume of 50 μL.

In the iEMS shaker (Thermo Scientific) with continuous stirring (1400rpm), the MPT reaction plate was incubated at 25°C for 10 minutes, and the reaction was terminated by adding 45 μL of Pi Blue stop reagent (PiBlue ^TM phosphate assay kit, POPB-DP, Bioassay Systems, the U.S.). The plate was mixed and sealed, then incubated at 25°C for 30 minutes in iEMS shaker (650rpm) to develop the color. After incubation, color formation was determined by measuring the absorbance at 620nm on a microplate reader (Spectramax, Molecular Device). The activity of each phytase sample to IP6 substrate was calculated based on the fitted standard curve of phytase protein, and the range of known concentration and activity was 0-350ppm, taking the mean value of three repetitions. The specific activity in μmol phosphate/mg/min was then calculated for each phytase sample using the activity at pH 3.5 or 5.5 divided by the protein concentration of phytase in the sample determined by RP-HPLC (as described in Example 2).

For the phytase variants described in Table 3B and Table 21, sample preparation and activity analysis were performed as described herein. An aliquot of the purified protein (Example 2) was diluted to a target concentration of 100 ppm in buffer (100 mM sodium acetate, 0.5 M NaCl pH 5.5), and then serially diluted to a final concentration of 0.1 ppm using 100 mM sodium acetate buffer, 0.025% Tween-20, and 0.05 mM CaCl ₂ (pH 5.5). The activity at pH 5.5 and 3.5 was then determined as described in Example 3, except that a 96-well MTP was used instead of a 384-well MTP (70 μL of IP6 substrate (0.20 mM) in 100 mM sodium acetate, 0.025% Tween 20, and 0.05 mM CaCl ₂ (pH 5.5) was reacted with a 10 μL aliquot of the diluted enzyme; the reaction was stopped using 170 μL of Pi Blue reagent).

Melting temperature (Tm) was determined by DSC.

Differential scanning calorimetry (DSC) measurements were performed using a MicroCal ^™ VP-capillary DSC system (GE Healthcare). DSC is a powerful analytical tool for characterizing the stability of proteins and other biomolecules. It measures the enthalpy (ΔH) and temperature (T _m ) of thermally induced structural transitions in solution. Phytase protein samples diluted to a final concentration of 0.4 mg/mL in 100 mM sodium acetate buffer (pH 5.5) were prepared. 400 μL of these protein samples and reference samples containing the same amount of protein-free buffer were added to a 96-well plate. The plate was placed in a temperature-controlled autosampler compartment maintained at 10°C. The protein samples and references were scanned from 20°C to 120°C at a scan rate of 2°C per minute. The melting temperature (Tm) was determined as the temperature at the maximum peak of the transition from the folded state to the unfolded state. The maximum change in Tm was ±0.2°C. The ORIGIN software package (MicroCal, GE Healthcare) was used for baseline subtraction and calculation of Tm values.

Example 4

Evaluation of specific activity and thermostability of phytase

(AB Vista公司)和

10000E(巴斯夫营养公司(BASF Nutrition))。选择这两种产品是因为它们是可商购的最固有的热稳定产品。表3A和3B提供了在pH 3.5和pH 5.5处以微摩尔磷酸盐/mg/min计的比活性的结果，以及通过DSC测量的以℃计的热稳定性(Tm)，其中ND表示值未确定。结果表明，所有高Tm植酸酶进化枝多肽及其片段均显示出远高于商业产品的Tm值。这些高Tm植酸酶进化枝多肽及其片段在pH 5.5处显示出与商业产品的比活性相当或比其更高的比活性。在pH 3.5处，高Tm植酸酶进化枝多肽及其片段的比活性均高于商业产品。在制粒条件下，尤其是在MLA中或以固体制剂应用时，较高的热稳定性是非常有益的。在单胃动物消化道中存在的酸性条件下，在酸性pH下较高的比活性可能是非常有益的。Using the method described in Example 3, samples of high Tm phytase clade polypeptides and fragments thereof produced using the methods described in Examples 1 and 2 were evaluated for phytase specific activity and thermostability at pH 3.5 and 5.5. The commercial phytase product Quantum

(AB Vista) and

10000E (BASF Nutrition). These two products were selected because they are the most inherently thermostable products commercially available. Tables 3A and 3B provide the results of specific activity in micromolar phosphate/mg/min at pH 3.5 and pH 5.5, as well as thermostability (Tm) in ° C measured by DSC, where ND indicates that the value is not determined. The results show that all high Tm phytase clade polypeptides and fragments thereof show Tm values far higher than commercial products. These high Tm phytase clade polypeptides and fragments thereof show specific activities comparable to or higher than the specific activities of commercial products at pH 5.5. At pH 3.5, the specific activities of high Tm phytase clade polypeptides and fragments thereof are all higher than commercial products. Under granulation conditions, especially in MLA or when applied in solid dosage forms, higher thermostability is very beneficial. Under acidic conditions present in the digestive tract of monogastric animals, higher specific activity at acidic pH may be very beneficial.

ND means value not determined

High resolution mass spectrometry (MS) was performed to confirm the amino acid sequences of the high Tm phytase clade polypeptides and fragments thereof: PHY-13594, PHY-11895, PHY-12663, PHY-13637, PHY-13789, PHY-13885, PHY-13936, PHY-14004, PHY-14256, and PHY-14277 (SEQ ID NOs: 1, 8, 11, 17, 22, 26, 27, 28, 30, 31). MS analysis confirmed the predicted C-terminus of SEQ ID NOs: 1, 8, 11, 17, 22, 26, 27, 28, 30, 31. In addition, MS analysis showed truncations of the N-terminus of SEQ ID NOs: 1, 8, 11, 17, 22, 26, 27, 28, 30, 31. The most commonly observed N-terminal amino acid corresponds to position 4 relative to the predicted mature sequence, but truncations are also observed at positions 2, 3, 5, 6, 7, 9, 10.

Example 5

Study on the Granulation Stability of Phytase

Feed pelleting recovery testing of high Tm phytase clade polypeptides and fragments thereof was performed in a pelleting facility at the Technological Institute (Sdr. Stenderup, Denmark). It should be noted that feed pelleting recovery depends on several factors, including: the specific feed matrix, conditioning and pelleting conditions, the assay used to determine activity, etc.

Blue 5G(AB Vista)和

E10000G(巴斯夫营养公司(BASF Nutrition))的进料制粒回收率。通过使用100mM乙酸钠缓冲液(pH 5.5)从粉末产品Quantum Blue 5G和Natuphos E10000G中提取植酸酶来获得参考商业植酸酶样品的液体样品。使用实例3中所述的参考植酸酶活性测定法(FTU)测量液体植酸酶样品的活性，并相应地给予至饲料中。根据以下程序，将PHY-11895、PHY-11932、PHY-12663、PHY-13637、PHY-13789、PHY-13885的液体样品应用到全谷物小麦载剂上，制成用于制粒稳定性研究的固体样品。将磨碎的全谷物小麦被转移到装有锯齿状刀片的切割搅拌器(coupe mixer)中。在混合的同时，将液体植酸酶样品(最多40％vol/w)添加到磨碎的全谷物小麦粉中。将液体植酸酶样品和磨碎的全谷物小麦粉的混合物放在托盘上，并在烘箱中于40℃干燥8-10小时。干燥后，使用Bühler研磨机(型号MLT 204)研磨固体植酸酶产品，并将辊隙设置为0。将参考商业产品样品Quantum Blue5G以固体(原样)给予至饲料中进行比较。使用参考植酸酶活性测定(FTU)进行了固体植酸酶产物的分析，并将产物相应地给予至饲料中。Phytases PHY-11895, PHY-11932, PHY-12663, PHY-13594, PHY-13637, PHY-13789, PHY-13885, PHY-13936, PHY-14256, PHY-14277 and reference commercial enzymes were measured

Blue 5G (AB Vista) and

Feed granulation recovery of E10000G (BASF Nutrition). Liquid samples of reference commercial phytase samples were obtained by extracting phytase from powder products Quantum Blue 5G and Natuphos E10000G using 100mM sodium acetate buffer (pH 5.5). The activity of the liquid phytase samples was measured using the reference phytase activity assay (FTU) described in Example 3 and administered to the feed accordingly. Liquid samples of PHY-11895, PHY-11932, PHY-12663, PHY-13637, PHY-13789, PHY-13885 were applied to whole grain wheat carriers to prepare solid samples for granulation stability studies according to the following procedure. The ground whole grain wheat was transferred to a cutting mixer (coupe mixer) equipped with a serrated blade. While mixing, the liquid phytase sample (up to 40% vol/w) was added to the ground whole grain wheat flour. The mixture of the liquid phytase sample and the ground whole grain wheat flour was placed on a tray and dried in an oven at 40°C for 8-10 hours. After drying, the solid phytase product was ground using a Bühler grinder (model MLT 204) and the roller gap was set to 0. A reference commercial product sample, Quantum Blue 5G, was given to the feed as a solid (as is) for comparison. The solid phytase product was analyzed using a reference phytase activity assay (FTU) and the product was given to the feed accordingly.

The feed composition was a corn/soybean diet comprising: 62.5% corn, 31% soy meal, 4.4% soy oil, 1.2% limestone, 0.5% VIT/MIN (Farmix Leghennen premix) and 0.4% sodium chloride. The moisture content of the feed was about 12%-14% (w/w). Between 120kg and 200kg of the above premixed feed was mixed with a liquid (MLA) or solid phytase sample for 10 minutes at room temperature (22°C-24°C) in a horizontal ribbon mixer to achieve a final phytase concentration of 5 FTU/g in the feed. The amount of liquid phytase sample added to the feed was between 0.2% and 0.5% (w/w).

模具和7.5kW马达的Simon Heesen压丸机中形成丸粒。调节进料螺杆速度以达到约300kg/小时的生产率，并且将辊速度设定为500rpm。所述系统可以在达到目标调节温度以预热制粒模具后运行约8分钟。随后收集5-7.5kg的压成丸粒的饲料样品，并立即在具有多孔底的冷却箱中冷却，伴随1500m3空气/h的环境空气流15分钟。在冷却期间，丸粒的水含量下降到与蒸汽调节之前含有植酸酶的饲料混合物(醪液饲料)相当的水平。根据ISO_6497_2002，使用分样器缩小样品的尺寸，并按如下来确定植酸酶的回收率。After mixing, the feed samples containing phytase were conditioned in a KAHL cascade mixer at 60°C, 80°C, 85°C, 90°C or 95°C for 30 seconds and then pressed into pellets. As used herein, the term "conditioning" means mixing the feed/enzyme mixture and treating it with steam to reach a target temperature of 60°C, 80°C, 85°C, 90°C or 95°C for a holding time of 30 seconds. The conditioning temperature was manually controlled by adjusting 3 steam valves, from which steam at a pressure of 2atm was introduced into the feed/enzyme mixture. The temperature was maintained at the target temperature +/- 0.3°C at the outlet of the regulator. This steam conditioning typically increases the water content of the feed by 2-5.5% by weight at a conditioning temperature of 60-95°C. Immediately after the conditioning step, the feed/enzyme mixture was placed in a steam mixer equipped with

The pellets were formed in a Simon Heesen pellet press with a 7.5kW motor and a 1.25 kW die. The feed screw speed was adjusted to reach a productivity of about 300 kg/hour, and the roller speed was set to 500 rpm. The system can run for about 8 minutes after reaching the target adjustment temperature to preheat the pelleting die. 5-7.5 kg of pelletized feed samples were subsequently collected and immediately cooled in a cooling box with a porous bottom, accompanied by an ambient air flow of 1500 m3 air/h for 15 minutes. During the cooling period, the water content of the pellets dropped to a level comparable to the feed mixture (mashed feed) containing phytase before steam conditioning. According to ISO_6497_2002, a sample divider was used to reduce the size of the sample, and the recovery of phytase was determined as follows.

Feed samples (both mash feed and pellets) containing phytase were ground using a Retch laboratory grinder (model ZM 200 equipped with a 0.75 mm screen) and then analyzed for phytase activity using the following method, which is a modification of the ISO 30024 procedure: "Animal feeding stuffs—Determination of phytase activity".

To extract phytase from feed samples, 20.0 g (+/- 0.05 g) of ground mash and pelleted feed were mixed with 100 mL extraction buffer (250 mM sodium acetate, 1 mM CaCl ₂ , 0.01% Tween-20, pH 5.5) on a rotary stirrer at room temperature for 20 minutes. The supernatant was collected after filtration (glass fiber filter, GA-55 from Advantec). The supernatant was further diluted in extraction buffer to obtain measurements within the linear range of the phosphate standard curve in the following FTU phytase assay (0.04 FTU/mL).

1 mL of the diluted phytase sample was mixed with 2 mL of 7.5 mM IP6 (sodium phytate from rice, Shanghai AZ Import and Export, Zhejiang Orient Phytic Acid Co. Ltd #Z0201301181) substrate solution in extraction buffer and incubated in a water bath at 37°C for 60 min. The reaction was terminated with 2 mL of acidic molybdate/vanadate reagent and the release of inorganic phosphate was quantified spectrophotometrically at 415 nm. The results were corrected by subtracting the absorbance of the sample corresponding to time zero (sample not incubated at 37°C for 60 min). A standard curve for phosphate was generated from dry potassium hydrogen phosphate and used to calculate the amount of phosphate released by each sample. One unit (FTU) is defined as the amount of phytase that produces 1 micromole of phosphate per minute. The feed pellet recovery percentage was calculated using the following formula: (phytase activity of pellets (FTU/g) divided by phytase activity of mash feed (FTU/g))*100.

Table 4 lists the feed pelleting recoveries percentages of the phytases shown in Table 3 applied to MLA at temperatures of 60°C, 80°C, 85°C, 90°C and 95°C. For all tested high Tm phytase clade polypeptides and fragments thereof applied to MLA, the pelleting recoveries at 95°C were at least 50%. For comparison, the extracted commercial reference phytases QuantumBlue and Natuphos E 10000 had much lower feed recoveries of 15% and 25%, respectively, under the same conditions. These data illustrate the high robustness of high Tm-phytase clade polypeptides and fragments thereof.

At 60°C, for all high Tm phytase clade polypeptides and fragments thereof tested, feed pelleting recoveries when applied to MLA were between 71% and 85%. It initially seemed contradictory that the robust phytases disclosed herein lost 15% to 29% of their activity when conditioned at 60°C and subsequently pressed into pellets when the 60°C conditioning temperature was more than 30°C lower than the Tm of the robust phytases. This suggests that the initial losses were not related to thermal inactivation in the conditioner, which is further confirmed by the fact that there were only limited additional losses when the temperature was raised to 80°C. Without being bound by theory, it is believed and has been described for other enzymes (phytases and xylanases; Test Report 875: Danish Agriculture & Food Council, Patent Application WO 2014120638 and Novus Insight, Issue 3, 2015), that there may be a portion of phytases and xylanases that are difficult to extract/recover from the feed after conditioning and pelleting. However, it is believed (again without being bound by theory) that at least a portion of this non-recoverable fraction is still biologically active in the animal - in other words, the non-recoverable fraction may not be irreversibly inactivated by heat stress. Instead, it is believed (again without being bound by theory) that the non-recoverable fraction is bound to the feed in a manner that cannot be extracted in vitro. Alternatively, it is believed (again without being bound by theory) that even if virtually all of the phytase protein is extracted, the activity of the phytase is significantly reduced when measured in an in vitro assay due to conditioning and pelleting. Without being bound by theory, it is believed that applying the phytase in MLA will increase the amount of this non-recoverable but biologically active form of the phytase compared to the dried and/or coated form due to direct physical interaction with the feed.

Therefore, a suitable way to assess the thermal robustness of phytases used in MLA is not to compare the recoverable activity in feed before and after conditioning and pelleting, but to compare the recovery of phytase activity after conditioning and pelleting at low (e.g. 80°C) and high (95°C) conditioning temperatures, which are within the commercially relevant conditioning temperature range.

ND means value not determined

Table 5 shows the ratio of feed pelleting recoveries when applied in MLA at 95°C for 30 seconds compared to 80°C for 30 seconds. For the high Tm phytase clade polypeptides and fragments thereof applied in MLA, the ratio of feed pelleting recoveries at 95°C compared to 80°C for 30 seconds in MLA ranged from 0.72 to 0.98. The corresponding value for the extracted commercial reference phytase Natuphos E 10000 was 0.32. The data indicate that the high Tm phytase clade polypeptides and fragments thereof disclosed herein are highly robust to changes in conditioning temperature within the commercially relevant temperature range.

*Ratio results are given for the 95°C process compared to the 85°C process (not 80°C).

ND means value not determined

Table 6 shows the feed pelleting recoveries at 95° C. for phytases PHY-11895, PHY-11932, PHY-12663, PHY-13637, PHY-13789, PHY-13885 and Quantum Blue 5G when applied as a solid. When applied as a solid to a ground whole grain wheat carrier, all high Tm phytase clade polypeptides and fragments thereof had a feed pelleting recovery of at least 64% at 95° C. The commercial powder product Quantum Blue 5G had a recovery of 58% when tested under the same conditions.

Example 6

In vivo evaluation of phytase

Performance Evaluation of PHY-12663, PHY-11932, and PHY-11895

The in vivo performance of phytases PHY-12663, PHY-11932 and PHY-11895 was evaluated in broilers. The study was conducted at Texas A&M University. Eight dietary treatments were tested: a positive control diet (PC) formulated to meet the nutritional requirements of broilers, a negative control (NC) formulated to lack digestible phosphorus (0.16% point lower than PC) and calcium (0.19% point lower than PC), and NC supplemented with PHY-12663, PHY-11932 or PHY-11895 phytases (doses of 500 and 1000 FTU/kg). Table 7 provides the dietary composition of the calculated and analyzed nutritional values used in this study.

Ground rice husk was used as phytase carrier.

One-day-old Cobb 500 male broilers were assigned to dietary treatments, each containing 10 replicates of 30 chicks per pen. The diet was based on corn, soybean meal and rice bran in the form of mash. On day 21, five chickens were taken from each replicate pen and tibiae were collected to determine fat-free tibia ash. The diet was prepared according to a 3-stage feeding program (starter days 0-10, grower days 11-21 and finisher days 22-42). Diet and water were available ad libitum during the 42-day study.

Data were analyzed using ANOVA, treatment means were separated using the Tukey HSD test, and P<0.05 was considered significant. The following performance parameters were calculated: ADG (average daily gain), ADFI (average daily feed intake), and FCR (feed conversion ratio) during the 0-42 day study period. Table 8 shows the growth performance results of broilers fed diets supplemented with different doses of different phytases during 0-42 days of age and tibia ash measured at 21 days of age. Compared with PC, the reduction of phosphorus (P) and calcium (Ca) in the NC diet was associated with lower ADG, ADFI, and tibia ash content and higher FCR. Compared with the NC diet, all tested phytases PHY-12663, PHY-11932, and PHY-11895 improved ADG, ADFI, tibia ash, and FCR in broilers. On average, treatment with PHY-12663, PHY-11932 and PHY-11895 phytases improved ADG by 12% and 14%, ADFI by 8% and 10%, FCR by 3.3% and 3.7%, and tibia ash by 9% and 9.7%, respectively, compared to the NC diet at doses of 500 and 1000 FTU/kg of feed. In addition, all phytases performed better or did not perform significantly differently than those fed the PC diet in all parameters. These data indicate that high Tm phytase clade polypeptides and their fragments (PHY-12663, PHY-11932 and PHY-11895) are able to significantly improve broiler bone growth and performance.

a, b, c: Different superscripts in the columns indicate significant differences, P < 0.05

FCR is the adjusted mortality rate

Performance evaluation of PHY-13789, PHY-13637, PHY-14004, and PHY-13885.

The in vivo performance of PHY-13789, PHY-13637, PHY-14004 and PHY-13885 was evaluated in broiler chickens at AH Pharma (Hebron, MD, USA). Ten treatments were tested, including a positive control diet (PC) formulated to meet the nutritional requirements of broiler chickens, and a negative control diet (NC). The NC diet was formulated to lack digestible phosphorus (no inorganic phosphate, 0.24% points lower than PC), calcium (0.19% points lower than PC), digestible AA (0.04%, 0.03% and 0.03% lower than PC for Lys, Met+Cys, Thr, respectively) and ME (69 kcal/kg lower than PC).

The performance of PHY-13789, PHY-13637, PHY-14004 and PHY-13885 was tested in the NC diet at two doses of 500 or 1000 FTU/kg feed. Male broiler chickens (Ross 308) were fed the same pre-starter diet from 0 to 5 days of age and received the test diet from 6 to 15 days of age. The dietary treatments were randomly assigned to nine cages/treatment with 8 birds in each cage. The diet was based on maize, wheat, soybean meal, rapeseed meal and rice bran (diet composition is shown in Table 9).

ND means value not determined

*ME: Metabolizable Energy

Body weight and feed intake were recorded on days 6 and 15. During the last 4 days, excreta were collected daily, weighed and pooled in one cage. The pooled excreta of each cage was used for the measurement of phosphorus (P) retention according to AOAC official method 965.17. On day 15, right tibiae of six chickens per cage were collected and used for the measurement of tibia ash.

Data were analyzed using ANOVA and treatment means were separated using the Tukey HSD test. P < 0.05 was considered statistically significant. Table 10 shows the effects on the performance of PHY-13789, PHY-13637, PHY-14004 and PHY-13885. Tibia ash was measured at 15 days of age and phosphorus retention was measured in broilers from 11 to 15 days of age. All tested phytases, PHY-13789, PHY-13637, PHY-14004 and PHY-13885, improved ADG, ADFI, tibia ash and phosphorus retention in broilers compared to NC.

a, b, c, ^etc .: Different superscripts in the columns indicate significant differences, P < 0.05

Phytase PHY-13789 improved ADG by 5.4% and 8.4%, FCR by 3.4% and 5.1%, tibia ash by 4.6% and 8.8%, and phosphorus retention by 15.3% and 35.4% at doses of 500 and 1000 FTU/kg, respectively, compared with NC. Phytase PHY-13637 improved ADG by 6.1% and 8.6%, FCR by 3.8% and 5.7%, tibia ash by 6.3% and 9.6%, and phosphorus retention by 17.7% and 35.8% at doses of 500 and 1000 FTU/kg, respectively, compared with NC. Phytase PHY-14004 improved ADG by 4.5% and 7.5%, FCR by 3.3% and 5%, tibia ash by 6.5% and 10.1%, and phosphorus retention by 19.3% and 36.4% at doses of 500 and 1000 FTU/kg, respectively, compared with NC. Phytase PHY-13885 improved ADG by 5.6% and 7.8%, FCR by 3.6% and 5.4%, tibia ash by 5.5% and 9.8%, and phosphorus retention by 17.7% and 36.9% at doses of 500 and 1000 FTU/kg, respectively, compared with NC. On average, high Tm phytase clade polypeptides and fragments thereof improved ADG by 5.4% and 8.1%, respectively, FCR by 3.5% and 5.3%, respectively, tibia ash by 5.7% and 9.6%, respectively, and phosphorus retention by 17.5% and 36.1%, respectively, compared to NC. Despite the large nutrient reduction of NC in this trial (total removal of inorganic phosphorus and reductions in Ca, digestible AA, and ME), all phytases tested at all doses did not perform significantly differently from PC on ADG, ADFI, and FCR. All phytases, except PHY-13789 and PHY-13885 at 500 FTU/kg, had similar tibia ash content to PC. Phosphorus retention (as a percentage of P intake) was improved in all phytase treatments compared to PC. Tibia ash and phosphorus retention results confirm that these phytases are effective in releasing phosphorus.

The results of both trials demonstrated that all high Tm phytase clade polypeptides and fragments thereof tested provided substantial improvements in animal performance.

Example 7

Identification of a novel clade of phytases

The peptide sequence and fragment thereof of the high Tm-phytase clade polypeptide shown in Example 3 are used to generate Hidden Markov Model (HMM) to identify sequence similarity.MUSCLE version 3.8.31 (MUSCLE:multiple sequence alignment with high accuracy and high throughput.[MUSCLE：High accuracy and high throughput multiple sequence alignment] R.C Edgar (20014) Nucleic Acid Res [nucleic acid research] 32:1792) is used for sequence alignment using default parameters. Subsequently, HMM builder software (builder software) HMMER version 3.1b1 (can be obtained from http://hmmer.org/) is used to generate HMM from multiple sequence alignment. Only two parameters have been used: prior probability (Priors)=none, and weight=none. Use the following command: /usr/bin/hmmbuild --pnone --wnone Variants_for_filing_draft_5.hmm Variants_for_filing_draft_5.fsa, where wnone = no relative weight (all sequences are assigned a uniform weight), and pnone = no prior probability is used, and the parameters are frequencies. All probability parameters are stored as negative natural logarithmic probabilities, with precision rounded to five decimal places to the right. For example, the probability is stored as 0:25log0:25=1:38629. The special case of zero probability is stored as the * symbol. Figure 1 (columns A to 1BB) shows the HMM probability scores for each position of the polypeptide sequence of the high Tm phytase clade phytase. The comprehensive score (COMP) of the HMM is shown in bold in the top 3 columns of Figure 1A. The position (P) and consensus sequence (C) of each amino acid are shown in the 1st column under P/C. The consensus high Tm phytase clade phytase polypeptide sequence was generated from the HMM shown in Figure 1 and is listed as SEQ ID NO:64.

Then, HMM is used to generate HMM sequence score for the global set of about 7000 unique phytases, including the phytase sequences available in public databases and patents. The rank of various sequences and the correlation of sequence score with thermostability (Tunfold, and Tm measured by DSC) (data not shown) are compared. Based on this analysis, novel high Tm phytase clade polypeptides all have HMM sequence scores greater than 1200, as shown in Table 11 for the phytases listed in Tables 3A and 3B.

版本10.2.4中使用MAFFT比对进行。基于此MAFFT序列比对，使用

版本10.2.4中的Geneious Tree Builder生成了显示序列关系的系统发生树，其如图2所示。High Tm phytase clade enzymes listed in Table 11: [PHY-13594 (SEQ ID NO: 1); PHY-10931 (SEQ ID NO: 2); PHY-10957 (SEQ ID NO: 3); PHY- 11569 (SEQ ID NO: 4); PHY-11658 (SEQ ID NO: 5); PHY-11673 (SEQ ID NO: 6); PHY-11680 (SEQ ID NO: 7); PHY-11895 (SEQ ID NO: 8); PHY-11932 (SEQ ID NO: 9); PHY-12058 (SEQ ID NO: 10); PHY-12663 (SEQ ID NO: 11); PHY-12784 (SEQ ID NO: 12); PHY-13177 (SEQ ID NO:13); PHY-13371 (SEQ ID NO:13); NO:14); PHY-13460 (SEQ ID NO:15); PHY-13513 (SEQ ID NO:16); PHY-13637 (SEQ ID NO:17); PHY-13705 (SEQ ID NO:18); PHY-13713 (SEQ ID NO:19); PHY-13747 (SEQ ID NO:20); PHY-13779 (SEQ ID NO:21); PHY-13789 (SEQ ID NO:22); PHY-13798 (SEQ ID NO:23 ); PHY-13868 (SEQ ID NO: 24); PHY-13883 (SEQ ID NO: 25); PHY-13885 (SEQ ID NO: 26); PHY-13936 (SEQ ID NO: 27); PHY-14004 ( SEQ ID NO:28); PHY-14215 (SEQ ID NO:29); PHY-14256 (SEQ ID NO:30); PHY-14277 (SEQ ID NO:31); PHY-14473 (SEQ ID NO:32); PHY -14614 (SEQ ID NO:33); PHY-14804 (SEQ ID NO:34); PHY-14945 (SEQ ID NO:35); PHY-15459 (SEQ ID NO:36); PHY-16513 (SEQ ID NO:37 )]; PHY-16812 (SEQ ID NO: 64); PHY-17403 (SEQ ID NO: 65); PHY-17336 (SEQ ID NO: 66); PHY-17225 (SEQ ID NO: 67); PHY-17186 (SEQ ID NO:68); PHY-17195 (SEQ ID NO:69); PHY-17124 (SEQ ID NO:70); PHY-17189 (SEQ ID NO:71); PHY-17218 (SEQ ID NO:72); PHY -17219 (SEQ ID NO:73); PHY-17204 (SEQ ID NO:74); PHY-17215 (SEQ ID NO:75); PHY-17201 (SEQ ID NO:76); PHY-17205 (SEQ ID NO :77); PHY-17224 (SEQ ID NO:78); PHY-17200 (SEQ ID NO:79); PHY-17198 (SEQ ID NO:80); PHY-17199 (SEQ ID NO:81); PHY-17214 (SEQ ID NO: 82); PHY-17197 (SEQ ID NO: 83); PHY-17228 (SEQ ID NO: 84); PHY-17229 (SEQ ID NO: 85); PHY-17152 (SEQ ID NO: 86); and PHY-17206 (SEQ ID NO: 87) and publicly disclosed microbial phytases: [Buciotella novosporium WP 064555343.1 (SEQ ID NO: 38); Citrobacter barkiii AAS45884.1 (SEQ ID NO: 39) ; Coxiellaceae RDH40465.1 (SEQ ID NO: 40); Enterobacteriaceae WP 094337278.1 (SEQ ID NO: 41); Escherichia coli WP001297112 (SEQ ID NO: 42); Hafnia alvei WP 072307456.1 (SEQ ID NO: 43); Rouxiella badensis WP 084912871.1 (SEQ ID NO: 44); Serratia species WP009636981.1 (SEQ ID NO: 45); Yersinia arnoldii WP004701026.1 (SEQ ID NO: 46 ); Yersinia freundii WP 050140790.1 (SEQ ID NO: 47); Yersinia kluyveri WP 004392102.1 (SEQ ID NO: 48); Yersinia moorei WP 049646723.1 (SEQ ID NO: 49); Yersinia rotheriae WP050539947.1 (SEQ ID NO: 50); EP3222714-0003APPM phytase (SEQ ID NO:51); US8101391-0002 (SEQ ID NO:52); US8101391-0004 (SEQ ID NO:53); US8101391-0035 (SEQ ID NO:54); US8101391-0049 (SEQ ID NO:55); US8143046- 0001 (SEQ ID NO:56); US8143046-0003 (SEQ ID NO:57); US8460656-0002 (SEQ ID NO:58); US8557555-0013 (SEQ ID NO:59); US8557555-0024 (SEQ ID NO:60 ); US20160083700-0003 (SEQ ID NO: 61); WO 2010034835-0002 (SEQ ID NO: 62)] was compared with the predicted mature sequence of

Based on this MAFFT sequence alignment, the

Geneious Tree Builder in version 10.2.4 generated a phylogenetic tree showing sequence relationships, which is shown in FIG2 .

Example 8

In vivo evaluation of phytase in chicken

This example evaluates the effect of a representative biosynthetic bacterial 6-phytase produced by a genetically engineered strain of Trichoderma reesei on ileal digestibility of shank ash and P (AID P) in broiler chickens when added to a basal diet reduced in Ca and P compared to a nutritionally adequate, non-phytase supplemented diet. In addition, feed intake, growth performance and feed conversion were observed.

Materials and methods

Experimental and Control Diets: A positive control (PC) diet based on corn and soybean meal was formulated to meet the nutritional requirements (P and Ca sufficiency) of chickens during the early (days 1–21) and late (days 22 to 42) phases [National Research Council, Nutrient Requirements of Poultry, 9th ed., Revised, Natl Acad Press, Washington, D.C. [DC]; 1994]. A negative control (NC) diet was formulated in which calcium (Ca) and available phosphorus (P) were reduced by 2.0 g/kg and 1.9 g/kg, respectively, in the early phase, and by 2.0 g/kg and 1.8 g/kg, respectively, in the late phase diets. See Table 12. Titanium dioxide (added at 4 g/kg) was included in all early phase diets as a marker of indigestibility. Negative control diets were tested as stand-alone diets or supplemented with 250, 500 or 1000 FTU/kg of a biosynthetic bacterial 6-phytase produced by a genetically engineered strain of T. reesei. Diets were available to the chickens ad libitum in the form of mash.

Chickens, broiler houses and experimental design: On the day of hatching, mixed-sex Cobb 500 broiler chickens (50% male, 50% female) were obtained from a commercial hatchery, which had been vaccinated against infectious bronchitis and Newcastle disease through drinking water at the hatchery. Vaccines against infectious bursal disease were also administered through drinking water on days 11-14. Chickens were allocated to floor pens based on initial body weight (BW), so that each pen contained chickens of approximately equal weight. A total of 1176 chickens were allocated to 49 pens, 24 birds per pen; NC was 9 pens, and all other treatments were 10 pens, each containing 50% males and 50% females, using a completely randomized design. The pens were located in an environmentally controlled broiler house with a lighting regimen of LD 18:6 and an initial temperature of 35°C, which was reduced to 24°C on day 28.

Sampling and measurements: Representative subsamples of all diets were analyzed for dry matter (DM), crude protein (CP), crude fat (CF), ash, P, potassium (K), magnesium (Mg), calcium, sodium (Na), phytate, and phytase.

Body weight and feed intake (FI) were measured on a pen basis on days 1, 21 and 42 and used to calculate BW, average daily gain (ADG), average daily feed intake (ADFI) and mortality-corrected feed conversion ratio (FCR). Mortality was checked and recorded daily.

On days 21 and 42, four birds (2 males, 2 females, sex determined at the sampling point) and six birds (3 males and 3 females) were randomly selected from each pen, respectively, and sacrificed with _CO2 gas and their left tibiae were collected and pooled (per pen) to determine defatted tibia ash. Ileal digesta were collected from euthanized birds on day 21, pooled per pen, and frozen on a Labconco FreeZone 12+ dehydrator (Labconco, Kansas City, MO). Dried feed and digesta samples were analyzed for P and Ca content to calculate nutrient digestibility using titanium dioxide as an inert marker.

Chemical Analysis: For all analyses, samples were analyzed in duplicate. Feed and ileal digesta were analyzed for nutrients according to the following methods: crude protein, NEN-EN-ISO 16634 [NEN-ISO 6492, en. Animal feedstuffs-Determination of fat content. International Organization for Standardization, Switzerland; 1999]; crude fat, NEN-ISO 6492 [NEN-ISO 6865, en. Animal feeding stuffs--Determination of crudefibre content--Method with intermediate filtration. International Organization for Standardization, Switzerland; 2000]; crude fiber, NEN-ISO 6865 [NEN-EN-ISO 16634, en. Animal Feeding Stuff-Determination of Nitrogen Content using Dumas combustion. International Organization for Standardization, Switzerland; 2000] Phosphorus, Ca, Mg, K and Na in feed and P and Ca in chyme were analyzed by microwave digestion and inductively coupled plasma optical emission spectrometry (OES) according to method AOAC 2011.14 [Official Methods of Analysis of AOAC International; 2011: Calcium, Copper, Iron, Magnesium, Manganese, Potassium, Phosphorus, Sodium, and Zinc in Fortified Food Products. Dietary phytate phosphorus (PP [inositol hexaphosphate (IP6)]) concentration and dietary phytase activity were determined by DuPont Laboratories (Brabrand, Denmark) using the method described by Yu et al. [Yu, S, Cowieson, A, Gilbert, C, Plumstead, P, Dalsgaard, S. Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin. J Anim Sci 2012;90:1824-1832]. One phytase unit (FTU) is defined as the amount of enzyme that liberates 1 μmol of inorganic orthophosphate per minute from a sodium phytate substrate at pH 5.5 and 37°C [AOAC International Method 2000.12: Phytase activity in feed: Colorimetric enzymatic method. Official Methods of Analysis of AOAC International. 17th ed.; Association of Official Analytical Chemists, Arlington, VA; 2000].

Tibia ash was measured using the following method: The fibula, muscle, and connective tissue were removed and the bones were dried at 100°C for at least 12 hours, then defatted in ether for 7-8 hours and air-dried. The defatted tibia was dried again at 100°C for at least 12 hours and then ashed in a ceramic crucible at 600°C for 24 hours.

Calculation: Feed conversion ratio (FCR) was calculated based on total BWG and total feed intake (corrected for mortality weight) for days 0-21, 22-42, and 0-42. Both ADG and AFDI were calculated by correcting for mortality, such as calculating ADFI from total feed intake for each period and dividing by the total number of days on feed. Mortality-corrected ADG was calculated by dividing mortality-corrected ADFI by mortality-corrected FCR.

The apparent ileal digestibility (AID, %) of P and Ca was calculated using titanium dioxide as an inert marker based on the following formula:

AID=1-[(Ti _d /Ti _i )×(N _i /N _d )]

Where _Tid is the titanium concentration in the diet, _Tii is the titanium concentration in the ileal digesta, _Ni is the concentration of the nutrient (P or Ca) in the ileal digesta, and _Nd is the concentration of the nutrient in the diet. All analytical values are expressed as g/kg dry matter.

Statistical analysis: Data are reported as the experimental unit of the column. Data were analyzed by analysis of variance (ANOVA) using the Fit Model platform of JMP 14.0 (SAS Institute Inc., Cary, NC, 1989-2019) to investigate the effects of treatments in the randomized design. Mean separation was achieved by Tukey's Honest Significant Difference test. Linear and quadratic responses with increasing phytase dose were analyzed using orthogonal polynomials. Differences were considered statistically significant at P < 0.05; P < 0.10 was considered a trend.

result

Diet Analysis: Phytase activity analyzed in the final diets confirmed the target dose levels (Table 13). The analytical values for CP in the basal (control) diet were within 10% of the calculated values. The reduction in P content achieved in the NC diet was well in line with the target reduction; total phosphorus content was reduced by 1.8 g/kg in the early diet and 2.3 g/kg in the late diet based on analytical values.

*The values stated are the average of the NC and NC+phytase treatments when making a batch of NC basal diet.

The analyzed phytase activities (FTU/kg) of PC, NC, NC+250FTU/kg, NC+500FTU/kg, and NC+1,000FTU/kg were 43, 24, 282, 480, 882 in the early stage, and <50, <50, 253, 594, 1110 in the late stage, respectively. Phytase activity in the diets was analyzed by DuPont Feed Technical Service, Brabrand, Denmark.

Nutrient digestibility: The AID of P was not significantly reduced in birds fed the NC diet relative to the PC diet (Table 14). Phytase supplementation increased the AID P relative to NC at dose levels of 500 FTU/kg or higher; and at 1000 FTU/kg, phytase improved the AID of P relative to PC (P<0.05). Phytase improved ileal digestible P (expressed on a g/kg basis) in the diets when given at 500 FTU/kg or higher (+1.39 g/kg relative to NC at 500 FTU/kg and +1.76 g/kg relative to NC at 1000 FTU/kg; P<0.05). At these dose levels, digestible P expressed in g/kg in the diets was comparable to that in the PC diet. The AID of Ca was not affected by dietary treatment, but increased linearly with increasing phytase dose from 0 to 1000 FTU/kg (P<0.10).

¹ Biosynthesis of bacterial 6-phytase produced by the genetically modified microorganism Trichoderma reesei.

² Increasing the phytase dose from 0 (NC) to 1,000 FTU/kg resulted in a linear and quadratic increase in AID P (P<0.05), while Ca showed an almost significant linear increase (P=0.052)

a, b, c: Least square means with different superscript letters in a row are different (P < 0.05, Tukey test).

Tibia ash: The effects of dietary treatment on tibia ash were highly significant (P<0.001) and are presented in Table 15. Birds fed the NC diet showed reduced tibia ash at d 21 and d 42 compared with PC (-6.7 and -4.1 percentage points, respectively; P<0.05). Phytase supplementation improved tibia ash sampled at d 21 and d 42 compared with NC at all three dose levels (P<0.05); tibia ash in all phytase treatments was comparable to PC.

^1All performance data are corrected for mortality

² Increasing the phytase dose from 0 (NC) to 1,000 FTU/kg resulted in linear and quadratic increases in all measured parameters (P < 0.05)

³ Biosynthesis of bacterial 6-phytase produced by the genetically modified microorganism Trichoderma reesei.

a, b, c: Least square means with different superscript letters in a row are different (P < 0.05, Tukey test).

Feed intake and growth performance: Table 15 also lists the effects of dietary treatment on feed intake, body weight and feed conversion rate. Treatment affected all response measures during all growth stages (early, late, and overall; P<0.01 in all cases). No significant differences in mortality were observed (data not shown).

Compared with PC, birds fed the NC diet showed decreased BW on days 21 and 42, increased FCR during the late stages and throughout the period, and decreased ADG and ADFI during all stages (P<0.05).

Supplementation of the experimental phytase at any dose level enabled the birds to overcome the P deficiency in the NC diet and had improved ADFI, BW, ADG and FCR during all phases (P<0.05), making them comparable to PC during all phases, regardless of phytase dose. The experimental phytase at a dose level of 1,000 FTU/kg resulted in birds with an average BW of 2.74 kg on day 42 and an average overall FCR of 1.626 (vs. 1.661 in PC).

In conclusion, this study shows that the experimental variant phytase, added to a formulated feed in which inorganic phosphorus from MCP was reduced by 1.8 to 1.9 g/kg and administered at dosage levels between 250 and 1000 FTU/kg, was effective in maintaining growth performance, tibia ash and ileal P digestibility, equivalent to a nutritionally adequate diet. The greatest beneficial effect was seen at 1000 FTU/kg. Based on the observed increase in digestible phosphorus, the estimated phosphorus replacement value from monocalcium phosphate was 1.64 and 2.07 g/kg diet (equal to 1.39 and 1.76 g/kg of digestible P from MCP) at 500 and 1000 FTU/kg, respectively.

Example 9

In vivo evaluation of porcine phytase

The aim of this study was to evaluate the efficacy of a diet supplemented with a representative experimental biosynthetic bacterial 6-phytase on bone ash and mineralization and growth performance in weaned pigs fed a corn-soybean meal-based diet to which inorganic phosphate was not added, compared to supplementation with inorganic P from MCP. For comparative purposes, an existing commercial phytase was included in the study. A secondary aim was to determine the digestible P equivalent value of the phytase in the test environment.

Materials and methods

PHY，杜邦营养生物科学公司(DuPont Nutrition and Biosciences))，在本文中称为PhyB。实验植酸酶是生物合成的细菌植酸酶，本文称为PhyX。PhyX由用真菌(里氏木霉)生产菌株发酵而产生，所述菌株表达通过祖先重建而组装的共有序列细菌植酸酶基因的生物合成变体，且对布丘氏菌属物种(杜邦营养生物科学公司(DuPont Nutrition andBiosciences))的序列有偏差。饮食以醪液形式可供仔猪随意摄取，并且水可自由获取，Experimental and control diets: A positive control (PC) diet based on corn and SBM was formulated to meet the nutritional requirements of piglets weighing 10 to 25 kg (NRC, 2012), containing 2.9 g/kg digestible P and 7.0 g/kg Ca (Table 16). A negative control (NC) diet was formulated without inorganic phosphate (1.1 g/kg digestible P) and with reduced Ca (5.0 g/kg). NC was tested as a standalone diet and was also supplemented with 500 or 1,000 FTU/kg commercial phytase diet, 250, 500 or 1,000 FTU/kg experimental phytase or 3 levels of MCP (+0.7, +1.4 and +1.8 g/kg digestible phosphorus from MCP) were added, equivalent to 1.8, 2.5 and 2.9 g/kg digestible P content (the latter constituted the PC diet). This resulted in a total of 9 dietary treatments. Additional limestone was added to the MCP-supplemented diet to maintain the Ca to P ratio in the range of 1.2 to 1.3 (Table 16). The commercial phytase was a microbial 6-phytase from Buttiauxella sp. expressed in Trichoderma reesei (

PHY, DuPont Nutrition and Biosciences), referred to herein as PhyB. The experimental phytase was a biosynthetic bacterial phytase, referred to herein as PhyX. PhyX was produced by fermentation with a fungal (Trichoderma reesei) production strain expressing a biosynthetic variant of a consensus sequence bacterial phytase gene assembled by ancestral reconstruction with a bias towards the sequence of Buttiauxella species (DuPont Nutrition and Biosciences). The diet was available ad libitum to the piglets in the form of mash, and water was freely available,

¹ Antioxidant, including BHT, propyl gallate and citric acid.

² Supplement per kg of diet: Iron (from FeSO ₄ ·H ₂ O), 120 mg; Iodine (from Ca(IO ₃ ) ₂ ) 0.75 mg; Cobalt (from 2CoCO ₃ ·3Co(OH) ₂ ·H ₂ O), 0.6 mg; Copper (from CuSO ₄ ·5H ₂ O), 6 mg; Manganese (from MnO) 60 mg; Zinc (from ZnO) 100 mg; Selenium (E8) (from Na ₂ SeO ₃ )0.37mg; Vitamin A, 10000UI; Vitamin D3, 2000UI; Vitamin E (α-tocopherol), 25mg; Vitamin B1, 1.5mg; Vitamin B2, 3.5mg; Vitamin B6, 2.4mg; Vitamin B12, 20μg; Vitamin K3, 1.5mg; Calcium pantothenate 14mg; Niacin, 20mg; Folic acid, 0.5mg; Biotin, 50μg.

^3. The test product was mixed with wheat vehicle to achieve the target dose, and the control treatment received vehicle only without the test product.

⁴ SID = standardized ileal digestibility.

Pigs, pig housing and experimental design: Experimental procedures complied with European Directive 2010/63/EU and Spanish guidelines for the care and use of animals in research (B.O.E. No. 252, Royal Decreto 2010/2005). A total of 162 crossbred Pietrain x (Large White x Landrace) 21-day-old piglets (initial body weight (BW) 6±1 kg) of mixed sex (50% male, 50% female) were obtained at weaning and fed a normal pre-adaptation diet until 42 days of age (~10-11 kg BW). The piglets were then grouped according to body weight and sex using a completely randomized block design and allocated to pens with 2 pigs per pen and 9 pens per treatment. The test diets were administered to pigs from 42 to 70 days of age. The pens were arranged together in an environmentally controlled animal room, where the temperature was initially maintained at 30°C and then decreased by 1°C per week.

Sampling and measurements: Representative subsamples of all diets were analyzed for dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), ash, minerals, phytate, and phytase.

Pigs were weighed before the start of the experiment and again on days 14 and 28 to calculate average daily gain (ADG). Feed disappearance was assessed on days 14 and 28 and used to calculate average daily feed intake (ADFI). Feed conversion ratio (FCR) was calculated from ADFI and ADG.

On day 28 of the experiment, one piglet per pen was euthanized by an intravenous overdose of sodium pentobarbital and the front and hind right feet were excised to determine metacarpal/metatarsal bone ash and mineralization (Ca and P). The feet were stored at -20°C until analysis.

Chemical analysis: All samples were analyzed in duplicate. Dry matter, ash, CP and ether extracts in the feed were analyzed according to AOAC (2000a) method (925.09, 942.05, 968.06 and 920.39, respectively). Nitrogen content was determined by Nitrogen FP-528 analyzer (Leco corp., St. Joseph, MO, USA) by Dumas procedure. Organic matter (OM) was calculated as the difference between DM and ash. Exogenous phytase activity in the feed was analyzed according to Engelen et al. (1994). One phytase unit (FTU) is defined as the amount of phytase that releases 1 mmol of inorganic phosphate per minute from 0.0051 mol/L sodium phytate at a standard pH of 5.5 and a temperature of 37°C (AOAC, 2000b).

Bone ash was determined on metacarpal III/IV and metatarsal III/IV from the right forefoot and right hindfoot, respectively. After extraction, the integrity of the bone was first characterized in a 3-point mechanical test using an Instron test system (Norwood, MA, US) with a model of 2519-106 equipped with a 2kN load cell. Biomechanical parameters such as extrinsic stiffness, ultimate force, displacement, and work of failure were used to characterize the integrity of the bone (Turner, 2006). The bone was then used to determine its DM content for 4 h in a 103 ° C oven, then burned in a 200 ° C oven for 3 h, then placed in a 550 ° C muffle furnace for 72 h and determined its ash content. The metacarpal bones were then ground using a pestle and mortar to obtain ash, which was then sent to the SCT laboratory (University of Lérida, Spain) for determination of minerals (Ca, P, Mg) by inductively coupled plasma mass spectrometry (ICP-MS; Agilent Technologies model 7700X) after sulfuric acid digestion. The mineral composition of the feed (Ti, Ca, P, Mg, Fe, Zn and Cu) was also analyzed on ash samples by ICP-MS at the SCT laboratory (Pacquette and Thompson, 2018).

Statistical analysis: Data were presented in pen units, except for bone ash and bone strength, which were presented in pig units. Data were analyzed by analysis of variance (ANOVA) using the Fit Model platform of JMP 14.0 (SAS Institute Inc., Cary, NC, 1989-2019) to investigate the effects of treatments in the randomized design. Mean separation was achieved by Tukey's Honest Significant Difference test. In addition, a two-way ANOVA was performed using the factors "phytase" (PhyG vs. PhyB) and dose (500 and 1000) to compare the two phytases at two dose levels (500 and 1000 FTU/kg). Linear and quadratic responses with increasing phytase dose were analyzed using orthogonal polynomials. In addition, linear regression was performed on metacarpal ash, ADG, and FCR according to the addition of increasing digestible P from MCP (e.g., NC, NC+0.7, NC+1.4, and NC+1.8 g/kg digestible P from MCP). Digestible P equivalents were calculated by applying the Y value at a given phytase dose and calculating the corresponding X value. Differences were considered significant at P < 0.05; P < 0.10 was considered a trend.

result

Diet Analysis: Analytical values of nutrient content in the diets are listed in Table 17. Phytase activity in the NC diet was ≤50 FTU/kg, indicating the absence of phytase cross contamination. The activities in the phytase supplemented diets were within 10% of the target value, except for treatments NC+PhyX 250 and NC+PhyX 500, where the activities were -20% and +27% relative to the target dose, respectively. The analyzed P content in the NC diet containing added P from MCP was close to the expected value based on the expected MCP addition level.

Table 17: Analyzed nutritional values of experimental diets

¹ Representative biosynthetic bacterial 6-phytase.

PHY，杜邦动物营养公司(DuPont Animal Nutrition))。 ² Microbial 6-phytase from Buttiauxella sp. expressed in Trichoderma reesei (

PHY, DuPont Animal Nutrition.

³ According to the AFZ-INRA table (Sauvant et al., 2002), metabolizable energy and net energy were calculated as 0.79 and 0.58 of gross energy and digestible energy, respectively.

⁴ Phytase activity in the diets was analyzed by DuPont Laboratories, Brabrand, Denmark.

Bone ash minerals and bone strength: At 70 days of age (experimental day 28), metacarpal bone ash, Ca, and P contents were reduced in piglets fed the basal NC diet relative to PC (P<0.05; Table 18). Supplementation with both phytases and at all dose levels improved bone ash and bone P content (%) compared with NC (P<0.05). Metacarpal bone ash and bone P contents were comparable to PC at 500 and 1,000 FTU/kg. Increasing the dose of PhyX from 0 (NC) to 1,000 FTU/kg resulted in linear and quadratic increases in metacarpal bone ash at day 28 (P<0.05). Phytase supplementation did not affect metacarpal bone Ca content. Metacarpal bone ash and P contents responded linearly with increasing levels of MCP-P in the diet (P<0.05). Metatarsal bone ash content showed the same response as metacarpal bone ash.

Table 18: Effects of increasing two phytase doses or inorganic P content on metatarsal and metacarpal ash and mineralization (% dry matter basis) and metacarpal strength in 70-day-old piglets

¹ Experimental biosynthesis of bacterial 6-phytase

PHY，杜邦营养生物科学公司(DuPont Nutrition and Biosciences))。 ² Commercial microbial 6-phytase from Buttiauxella sp. expressed in Trichoderma reesei (

PHY, DuPont Nutrition and Biosciences).

The effects of dietary treatments on metacarpal biomechanical parameters are shown in Table 18. Ultimate force (N) was lower in NC compared to all other treatments (P<0.05). All phytase treatments at all dose levels improved ultimate force compared to NC. Both phytases maintained the same ultimate force at 1000 FTU/kg relative to PC. Compared to PC containing an additional 1.8 g of digestible P from MCP per kg of diet, both phytases improved stiffness (mPa) relative to NC at 500 FTU and 1000 FTU, and maintained stiffness (mPa) and work of destruction (J) at 1000 FTU/kg. When comparing the two dose levels of the two phytases, phytases at 1000 FTU/kg showed higher bone ash, ultimate force (N), stiffness (mPa), and work of destruction (J) compared to phytases at 500 FTU/kg (P<0.05). No interaction was found between phytase source and dose level.

Growth performance: The effects of dietary treatments on growth performance are shown in Table 19. With the exception of ADFI during d 0–14 (trend, P = 0.08), all growth performance response measures were impaired (reduced ADG and ADFI; increased FCR) in piglets fed the PC diet compared with those fed the NC diet (P < 0.05).

During the first phase of the experiment (days 0–14), 1,000 FTU/kg of PhyX and PhyB produced greater ADG and reduced FCR relative to NC (P<0.05) and were equivalent to PC containing 1.8 g/kg of added P from MCP.

During the second phase of the experiment (days 15 to 28), PhyX improved ADG relative to NC at 250 FTU/kg or higher, and improved FCR relative to NC at 500 FTU/kg or higher (P<0.05). PhyB also improved ADG and FCR relative to NC at both dose levels (P<0.05). At 500 FTU/kg or higher, both phytases produced ADG and FCR values comparable to PC containing 1.8 g/kg of added P from MCP.

Table 19: Effects of increasing two phytases or inorganic P levels on the performance of weaned piglets (42 to 70 days of age).

NC + digestible P from MCP (g/kg)

¹ Representative biosynthetic bacteria 6-phytase

PHY，杜邦营养生物科学公司(DuPont Nutrition and Biosciences))。 ² Commercial 6-phytase from Buttiauxella sp. expressed in Trichoderma reesei (

PHY, DuPont Nutrition and Biosciences).

³ Increasing the dose of PhyX from 0 (NC) to 1,000 FTU/kg resulted in linear and quadratic increases in ADG and FCR throughout the entire period (days 0-28) (P<0.05).

a, b: Least square means with different superscript letters in a row are different (P < 0.05, Tukey test).

Throughout the period (days 0–28), both phytases improved ADG relative to NC at all doses, and both phytases at 500 FTU/kg or above improved FCR relative to NC (P<0.05). At 500 FTU/kg or higher, either phytases produced ADG and FCR values comparable to PC containing 1.8 g/kg of added P from MCP. In addition, increasing PhyX dose from 0 to 1,000 FTU/kg resulted in linear and quadratic increases in ADG and decreases in FCR throughout the period (P<0.05). ADG and FCR responded linearly with increasing levels of MCP-P in the diet (P<0.05). Comparing the two dose levels of both phytases, FCR was lower at 1,000 FTU/kg compared with 500 FTU/kg (1.59 vs. 1.66, P<0.05). At 1000 FTU/kg vs. 500 FTU/kg, a trend toward increased ADG was observed (635 vs. 590, P=0.08), with no difference in feed intake (data not shown). No interaction was found between phytase source and dose level.

[00136] Inorganic P equivalents: Dietary digestible P equivalent values (g/kg diet) for PhyX and PhyB were calculated based on bone ash, ADG, and FCR as response parameters, using the observed responses to increasing digestible P from MCP as reference. The response to increasing digestible P from MCP was linear and positive for all three response measures (P < 0.001; Table 20). The calculated digestible P equivalent values increased with increasing phytase dose, regardless of the response parameter used, and reached a maximum of 1,000 FTU/kg (Table 20). At this dose level, PhyX had higher digestible P equivalent values than PhyB (mean values of the response parameters were 1.83 g/kg and 1.66 g/kg, respectively), with ADG being the highest and bone ash being the lowest (as a response parameter).

Table 20: Linear regression analysis of bone ash, ADG, and FCR responses to increasing digestibility from ^MCP1,2

¹ Linear regression was performed with increasing added digestible P from MCP (e.g., NC, NC+0.7, NC+1.4, and NC+1.8 g/kg digestible P from MCP) versus metacarpal ash, ADG, and FCR with the equation Y=a+bX, where Y is the response parameter and X is the increasing added digestible P from MCP.

² R ² based on regression of treatment means. Digestible P equivalents were calculated by applying the response parameter (Y, e.g. bone ash) value at a given phytase dose and calculating the corresponding MCP-P substitution (X) value.

In conclusion, this study showed that an experimental phytase (PhyX) was effective in maintaining metacarpal bone ash, bone P content, and growth performance in piglets when added at dose levels of 500 or 1,000 FTU/kg to a corn-soybean meal-based diet to which inorganic P was not added, equivalent to a nutritionally adequate diet (containing 2.9 g/kg digestible P, of which 1.8 g/kg was from MCP). The response was greatest at a dose level of 1,000 FTU/kg, at which it was estimated that the experimental phytase could replace an estimated 1.83 g/kg of digestible P in the diet in weaned pigs fed a corn-SBM-based diet containing rice and rice bran.

Example 10

Design and evaluation of chimeric high-Tm phytase clade peptides

A series of chimeric polypeptides were designed to evaluate the contribution of the exchange/replacement regions at the N-terminus and/or C-terminus of the high Tm phytase clade polypeptides (PHY-13594, PHY-13789, and PHY-13885) described in Example 4. For the purposes of this study, the N-terminus was defined as residues 1-13 according to SEQ ID NO: 1, the core region was defined as residues 14-325 according to SEQ ID NO: 1, and the C-terminus was defined as residues 326 according to SEQ ID NO: 1 to the end of each polypeptide described in Example 7. Proteins were produced using the methods described in Example 1, and clarified culture supernatant samples were used to measure thermal stability and phytase activity at pH 3.5 and pH 5.5 by DSC using the methods described in Example 3. PHY-13594, PHY-13789, and PHY-13885 phytases were used for comparison and the effects of chimeric molecules containing the N-terminal region of HAP phytases found in Buttiauxella sp. (Buttiauxella NCIMB 41248, SEQ ID NO:88), Citrobacter braakii (Citrobacter braakii AAS45884, SEQ ID NO:89), and E. piscicida (E. piscicida YP007628727, E. piscicida WP_015461291.1, SEQ ID NO:90). Likewise, PHY-13594, PHY-13789, and PHY-13885 were used for comparison to generate the effects of chimeric molecules containing the C-terminal regions of HAP phytases found in Hafnia alvei (Hafnia alvei WO 2010034835-0002, SEQ ID NO:94), Yersinia morganii (Yersinia morganii WP032813045, SEQ ID NO:95), and Buttiauxella sp. (Buttiauxella NCIMB 41248, SEQ ID NO:96). The phytase core regions used were as follows: SEQ ID NO:100 for PHY-13594, SEQ ID NO:101 for PHY-13789, and SEQ ID NO:102 for PHY-13885. Table 21 has described the various chimeric constructs tested, and provides the results of thermostability, specific activity at pH 3.5 places and the ratio of specific activity at pH 3.5 and pH 5.5 places.As shown in Table 21, the modification of the N-terminal or C-terminal of the three high Tm phytases assessed causes the enzyme with very similar thermostability, shows that the structural determinant cluster that is used to maintain the thermostability of these high Tm phytases is located in the amino acid sequence of the core region.When the mensuration described in the use example 2 was tested, all high Tm phytase clade polypeptides described in the table 21 also demonstrated greater than 100 FTU/mg.

For illustration, Figure 3 depicts the three-dimensional structure of a representative high Tm clade phytase, modeled using the published crystal structure of the closely related Hafnia alvei 6-phytase (PDB entry code: 4ARO, phytase in complex with inositol hexadecyl sulfate) and displayed as a ribbon diagram. The model was constructed using MOE (v2013.08, Chemical Computing Group Inc.) and visualized using the PyMol software program (version 1.8.4.2, Schrodinger, LLC). The "core" domain is represented in black, and the light grey tones are the N- and C-terminal regions that have been replaced/exchanged in the experiments shown herein. This model is consistent with the structure-based multiple sequence alignment proposed by Ariza et al. using the crystal structure of the 6-phytase from Hafnia alvei (Degradation of Phytate by the 6-Phytase from Hafnia alvei: A Combined Structural and Solution Study, PLOS, 8:1-13).

Sequence Listing

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Arg Glu Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala

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Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

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Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

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Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

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His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

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Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

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Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

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Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

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Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Leu Pro

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35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Trp Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

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Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His

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Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

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Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Gln His Ser Val Glu Lys Pro Cys Asp Phe Ala

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Gln Met Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val

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His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

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Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Arg Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

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Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Leu Pro

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Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

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85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Pro Leu

100 105 110

Thr Ile His His Gln Ser Asn Ile Lys Val Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Thr Gly Leu Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp Lys

145 150 155 160

Tyr Ala Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Gln His Ser Val Glu Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Lys Leu Ser Ile Gly Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Val Thr Phe Thr Val

385 390 395 400

Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 7

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-11680

<400> 7

Ser Glu Thr Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asn Ile Lys Val Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His

145 150 155 160

Tyr Arg Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Thr Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Lys Leu Ile Ile Thr Asp Asp Gly Asn Lys Val

195 200 205

Ala Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Lys Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro

405 410

<210> 8

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-11895

<400> 8

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Leu Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Trp Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Lys Asn Ile Lys Val Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Ala Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Thr Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Thr Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 9

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-11932

<400> 9

Ser Glu Thr Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Ala Glu Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His

145 150 155 160

Tyr Arg Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Ala Arg His Ser Gly Asp Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Met Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 10

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-12058

<400> 10

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Lys Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Pro Leu

100 105 110

Thr Ile His His Gln Ser Asn Ile Lys Val Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ser Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Ala Gln His Ser Gly Glu Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Ser Val Glu Pro Ala Cys Gln Ile Pro

405 410

<210> 11

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-12663

<400> 11

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Trp Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu

100 105 110

Thr Ile His His Gln Lys Asn Ile Lys Val Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Thr Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Asp Glu Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Arg Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro

405 410

<210> 12

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-12784

<400> 12

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Leu Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Asn Gln Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Ile Leu Ser Lys Asp Ser Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Met Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln Tyr

145 150 155 160

Tyr Ala Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Thr Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Thr Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Thr Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Val Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro

405 410

<210> 13

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13177

<400> 13

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Asn Gln Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Ala Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Arg Cys Asp Leu

100 105 110

Thr Ile His His Gln Lys Asp Ile Lys Gln Val Asp Pro Met Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Ala Gln His Ser Gly Glu Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Arg Trp Arg Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 14

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13371

<400> 14

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Gly Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asn Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ala Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Ile Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 15

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13460

<400> 15

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Ala Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Val His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asn Lys Ser Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Met Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Met Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 16

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13513

<400> 16

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Gly Pro Ile Glu Thr Leu Asn Gln Arg

145 150 155 160

Tyr Gln Ala Glu Leu Ala Leu Met Ser Ser Val Leu Asp Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Val Asp Lys Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 17

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13637

<400> 17

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Ser Val Leu Asp Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln His His Ser Gly Asp Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Val Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 18

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13705

<400> 18

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Arg Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Arg Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 19

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13713

<400> 19

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Thr Pro Asp Ala Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile His Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Val Asp Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Met Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 20

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13747

<400> 20

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Asp Leu

100 105 110

Ala Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Met Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Glu Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Met Gln Thr Ile Ser Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile

405 410

<210> 21

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13779

<400> 21

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro His Gln Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Glu Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile

405 410

<210> 22

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13789

<400> 22

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 23

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13798

<400> 23

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Ala Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asn Ile Ser Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Met Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asn Pro Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 24

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13868

<400> 24

Ser Asp Thr Ala Pro Ala Gly Phe Gln Leu Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Ser Gln Thr Tyr Glu

130 135 140

Ala Val Glu Lys Gln Ala Gly Gly Pro Ile Glu Thr Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Arg His Ser Gly Asp Lys Thr Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Glu Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 25

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13883

<400> 25

Ser Glu Ala Ala Pro Ser Gly Tyr Gln Leu Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asn Ile Ser Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

His Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 26

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13885

<400> 26

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 27

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-13936

<400> 27

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Pro Arg Asp Ser Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Ala Ile

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Glu Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 28

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14004

<400> 28

Ala Glu Glu Ala Asn Gly Met Lys Leu Gln Lys Ala Val Ile Leu Ser

1 5 10 15

Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg Asp

20 25 30

Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr Ile

35 40 45

Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr Arg

50 55 60

Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro Thr

65 70 75 80

Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg Lys

85 90 95

Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu Thr

100 105 110

Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His Pro

115 120 125

Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln Ala

130 135 140

Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His Tyr

145 150 155 160

Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys Ser

165 170 175

Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala Asn

180 185 190

Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val Gln

195 200 205

Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe Leu

210 215 220

Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile His

225 230 235 240

Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln Phe

245 250 255

Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr Pro

260 265 270

Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala Arg

275 280 285

Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala Gly

290 295 300

His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Thr Trp

305 310 315 320

Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu

325 330 335

Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val His

340 345 350

Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu Thr

355 360 365

Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys Asp

370 375 380

Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg Leu

385 390 395 400

Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 29

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14215

<400> 29

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu

100 105 110

Ala Ile His His Gln Gln Asn Ile Thr Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Arg His Ser Gly Asp Lys Thr Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Lys Val

195 200 205

Ala Leu Asp Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Ser Gln Trp Lys Ser Leu Leu Asn Leu His Asn Ala His

245 250 255

Phe Asn Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Arg Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Glu Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 30

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14256

<400> 30

Ser Asp Thr Ala Pro Ala Gly Tyr Gln Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Val Asp Lys Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Gln Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 31

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14277

<400> 31

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Pro Lys Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Arg His Ser Gly Asp Lys Thr Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile

405 410

<210> 32

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14473

<400> 32

Ala Glu Glu Gln Asn Gly Met Lys Leu Gln Lys Ala Val Ile Leu Ser

1 5 10 15

Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg Asp

20 25 30

Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr Ile

35 40 45

Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr Arg

50 55 60

Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro Thr

65 70 75 80

Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg Lys

85 90 95

Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Ala Ile Thr

100 105 110

Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His Pro

115 120 125

Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln Ala

130 135 140

Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His Tyr

145 150 155 160

Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys Ser

165 170 175

Pro Tyr Cys Arg His Gln Ser Gly Asp Lys Thr Cys Asp Phe Ala Asn

180 185 190

Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val Gln

195 200 205

Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe Leu

210 215 220

Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile His

225 230 235 240

Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln Phe

245 250 255

Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr Pro

260 265 270

Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu Ser

275 280 285

Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala Gly

290 295 300

His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr Trp

305 310 315 320

Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu

325 330 335

Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val Lys

340 345 350

Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu Thr

355 360 365

Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys Asp

370 375 380

Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg Leu

385 390 395 400

Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 33

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14614

<400> 33

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gly Cys Asp Leu

100 105 110

Thr Ile His His Gln Gln Asn Ile Thr Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Gln His Ser Val Asp Lys Pro Cys Asp Phe Ala

180 185 190

Asn Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 34

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14804

<400> 34

Ser Asp Thr Ala Pro Ala Gly Phe Gln Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Pro Arg Asp Asn Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Glu Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 35

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-14945

<400> 35

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Glu Arg Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Ala Ile

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 36

<211> 412

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-15459

<400> 36

Ala Glu Pro Gln Asn Gly Met Lys Leu Asp Lys Ala Val Ile Leu Ser

1 5 10 15

Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg Asp

20 25 30

Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr Ile

35 40 45

Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr Arg

50 55 60

Gln Tyr Phe Gln Gln Gln Gly Ile Leu Ser Lys Asp Arg Cys Pro Arg

65 70 75 80

Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg Lys

85 90 95

Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Pro Leu Thr

100 105 110

Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His Pro

115 120 125

Val Lys Gly Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln Ala

130 135 140

Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His Tyr

145 150 155 160

Arg Pro Glu Leu Ala Leu Met Ser Ala Val Leu Asn Phe Pro Ala Ser

165 170 175

Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala Gln

180 185 190

Met Met Pro Ser Lys Leu Tyr Ile Thr Asp Asp Gly Asn Glu Val Gln

195 200 205

Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe Leu

210 215 220

Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile His

225 230 235 240

Ser Glu Gln Glu Trp Asn Ala Leu Leu Lys Leu His Asn Ala Tyr Phe

245 250 255

Asp Leu Met Tyr Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr Pro

260 265 270

Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala Arg

275 280 285

Glu Leu Pro Asp Ile Ser Pro Asp Asn Arg Ile Leu Phe Leu Ala Gly

290 295 300

His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp Trp

305 310 315 320

Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu

325 330 335

Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val His

340 345 350

Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu Thr

355 360 365

Leu Gln Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Ser Cys Asp

370 375 380

Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Lys Leu

385 390 395 400

Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro

405 410

<210> 37

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> PHY-16513

<400> 37

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Met Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Asn Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Arg Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Met Phe His

115 120 125

Pro Val Lys Gly Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Asp His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Ala Val Leu Asn Phe Pro Ala

165 170 175

Ser Pro Tyr Cys Gln Gln His Ser Val Asp Lys Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Lys Leu Asn Ile Thr Asp Asp Gly Asn Glu Val

195 200 205

Ala Leu Asp Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Asn Glu Ser Gln Trp Lys Ser Leu Leu Asn Leu His Asn Ala His

245 250 255

Phe Asn Leu Met His Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Asn Glu Pro Ala Gly Thr Val Pro Leu Lys Ile Pro Ser Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Val

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Ile Pro

405 410

<210> 38

<211> 413

<212> PRT

<213> Buttiauxella noackiae

<400> 38

Ser Glu Thr Pro Ala Ser Gly Tyr Gln Val Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asn Thr Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Glu Lys Phe Gln Gln Gln Gly Ile Leu Ser Gln Glu Asn Cys Pro

65 70 75 80

Ala Pro Asn Ser Val Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Leu

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Gly Leu

100 105 110

Thr Ile His His Gln Gln Asn Leu Glu Lys Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Glu Lys Asn Gln Ile Gln Gln

130 135 140

Ala Val Glu Lys Glu Ala Gln Thr Pro Ile Asp Asn Leu Asn Gln His

145 150 155 160

Tyr Leu Pro Ser Leu Ala Leu Met Asn Thr Thr Leu Asn Phe Ala Asn

165 170 175

Ser Ala Trp Cys Gln Lys His Ser Val Asp Lys Ser Cys Asp Leu Gly

180 185 190

Gln Ser Met Pro Ser Lys Leu Ser Ile Gln Glu Asn Gly Asn Lys Ile

195 200 205

Thr Leu Asp Gly Ala Ile Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Lys Ala Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Ala Ala Leu Leu Lys Leu His Asn Thr Gln

245 250 255

Phe Asp Leu Met Ala Arg Thr Pro Tyr Ile Ala Arg His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asp Pro Asn Ala Thr Ala

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ser Gly Met Leu Asn Ile Ser

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Leu Ala Asp Lys Ala Gly Lys Gln Tyr Val Ser Val

340 345 350

Ser Met Val Tyr Gln Thr Leu Glu Gln Leu His Ala Gln Thr Pro Leu

355 360 365

Ser Leu Lys Glu Pro Ala Gly Ser Val Gln Leu Lys Ile Pro Gly Cys

370 375 380

Ser Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Pro Thr Phe Thr Arg

385 390 395 400

Val Val Ser Gln Ser Val Glu Pro Gly Cys Gln Leu Gln

405 410

<210> 39

<211> 413

<212> PRT

<213> Citrobacter braakii

<400> 39

His Ala Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val Val Ile Val

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys

20 25 30

Asp Val Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp

35 40 45

Leu Thr Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln

50 55 60

Arg Leu Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro

65 70 75 80

Ser Pro Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile

100 105 110

Gln Val His Tyr Gln Lys Asp Glu Glu Lys Asn Asp Pro Leu Phe Asn

115 120 125

Pro Val Lys Met Gly Lys Cys Ser Phe Asn Thr Leu Lys Val Lys Asn

130 135 140

Ala Ile Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg

145 150 155 160

Tyr Gln Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln

165 170 175

Ser Glu Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro

180 185 190

Glu Ala Leu Pro Ser Glu Phe Lys Val Thr Pro Asp Asn Val Ser Leu

195 200 205

Pro Gly Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu

210 215 220

Gln Glu Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly

225 230 235 240

Glu Lys Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp

245 250 255

Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu

260 265 270

Leu Asp Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn

275 280 285

Arg Tyr Gly Ile Lys Leu Pro Val Ser Leu Leu Phe Ile Ala Gly His

290 295 300

Asp Thr Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Lys Trp Ser

305 310 315 320

Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe

325 330 335

Glu Lys Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser

340 345 350

Phe Val Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser

355 360 365

Leu Glu Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu

370 375 380

Glu Lys Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu

385 390 395 400

Ile Lys Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

405 410

<210> 40

<211> 415

<212> PRT

<213> Coxiellaceae bacteria

<400> 40

Val Val Ala Lys Gly Thr Glu Tyr Thr Leu Gln Gln Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ser Pro Ile Lys His Ser Lys Leu Leu Asn

20 25 30

Glu Ile Thr Pro Ser Ser Trp Pro Gln Trp Pro Val Lys Pro Gly Tyr

35 40 45

Leu Thr Pro Arg Gly Lys Leu Leu Met Thr Leu Met Gly Glu Phe Tyr

50 55 60

Gly Asp Tyr Phe Arg Asn Lys Gly Leu Leu Ala Glu His Gly Cys Pro

65 70 75 80

Ala Asn Gly Thr Val Tyr Val Gln Thr Asp Val Asp Gln Arg Thr Ile

85 90 95

Leu Ser Gly Trp Ala Leu Leu Ser Gly Met Thr Pro His Cys Arg Phe

100 105 110

Lys Ile His His Gln Glu Asn Leu Lys Arg Ile Asp Pro Leu Phe His

115 120 125

Pro Val Glu Ala Gly Ile Cys Glu Leu Asn Lys Glu Lys Ala Leu Asn

130 135 140

Ala Ile Glu Glu Arg Leu Gly Ala Pro Leu Gln Thr Leu Ser Lys Arg

145 150 155 160

Tyr Ala Ser Pro Leu Ala Gln Met Ser Lys Ile Leu Lys Phe Asp Arg

165 170 175

Ser Pro Tyr Cys Glu Lys Met His Lys Met Gln Lys Ser Cys Asp Phe

180 185 190

Ala Thr Phe Leu Ser Asn Lys Ile Tyr Ile Asn Asn Lys Gly Thr Ile

195 200 205

Leu Leu Arg Gly Pro Val Ser Leu Ser Ser Thr Phe Ala Glu Ile Phe

210 215 220

Leu Leu Gln Asn Ser Gln Gly Met Pro Asp Val Ala Trp His Arg Leu

225 230 235 240

Lys Gly Glu Ala Asn Trp Glu Ser Leu Leu Ser Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ala Lys Thr Phe Tyr Ile Ser Arg His Glu Gly Thr

260 265 270

Pro Leu Leu Glu Glu Ile Gly Gly Ala Leu Thr His Gln Met Lys Gln

275 280 285

Gln Arg Ile Phe Ser Thr Leu Pro Leu Ser Ala His Asn Arg Val Leu

290 295 300

Phe Leu Val Gly His Asp Val Asn Ile Ala Asn Ile Ala Gly Met Leu

305 310 315 320

Gly Leu Asn Trp Gln Leu Leu Gln Gln Pro Asp Asn Thr Pro Pro Gly

325 330 335

Gly Gly Leu Val Phe Glu Leu Trp Gln Lys Met Asp Asp His Lys His

340 345 350

Tyr Ile Ser Ile Lys Met Phe Tyr Gln Thr Met Val Gln Leu Arg Asn

355 360 365

Lys Gln Lys Met Asp Leu Trp Leu Asn Pro Ala Gly Met Leu Ser Ile

370 375 380

Pro Gly Cys Asp Asn Met Gly Lys Asp Lys Leu Cys Arg Leu Glu Lys

385 390 395 400

Phe Gln Lys Lys Leu Gln Gln Ala Ile Glu Pro Ile Cys Arg Ile

405 410 415

<210> 41

<211> 411

<212> PRT

<213> Unknown sequence

<220>

<223> Enterobacteriaceae WP 094337278.1

<400> 41

Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser

1 5 10 15

Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp

20 25 30

Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu

35 40 45

Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg

50 55 60

Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln

65 70 75 80

Pro Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys

85 90 95

Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr

100 105 110

Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro

115 120 125

Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala

130 135 140

Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg

145 150 155 160

Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser

165 170 175

Asn Leu Cys Leu Asn Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr

180 185 190

Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu

195 200 205

Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu

210 215 220

Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp

225 230 235 240

Ser His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr

245 250 255

Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu

260 265 270

Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His Pro Pro Gln Lys Gln

275 280 285

Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His

290 295 300

Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr

305 310 315 320

Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe

325 330 335

Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser

340 345 350

Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser

355 360 365

Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu

370 375 380

Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile

385 390 395 400

Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu

405 410

<210> 42

<211> 411

<212> PRT

<213> Escherichia coli

<400> 42

Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser

1 5 10 15

Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp

20 25 30

Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu

35 40 45

Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg

50 55 60

Gln Arg Leu Val Ala Asp Gly Leu Leu Ala Lys Lys Gly Cys Pro Gln

65 70 75 80

Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys

85 90 95

Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr

100 105 110

Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro

115 120 125

Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala

130 135 140

Ile Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg

145 150 155 160

Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser

165 170 175

Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr

180 185 190

Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu

195 200 205

Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu

210 215 220

Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp

225 230 235 240

Ser His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr

245 250 255

Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu

260 265 270

Leu Asp Leu Ile Met Ala Ala Leu Thr Pro His Pro Pro Gln Lys Gln

275 280 285

Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His

290 295 300

Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr

305 310 315 320

Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe

325 330 335

Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser

340 345 350

Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser

355 360 365

Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu

370 375 380

Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile

385 390 395 400

Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu

405 410

<210> 43

<211> 411

<212> PRT

<213> Hafnia alvei

<400> 43

Ser Glu Thr Val Pro Ser Gly Tyr Gln Leu Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asp Ala Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Thr Phe Gln Gln Leu Gly Ile Leu Ser Lys Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys His Leu

100 105 110

Ser Ile His His Gln Gln Asp Ile Lys Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Val Cys Ser Met Glu Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Gln Gln Ala Gly Met Pro Ile Ala Gln Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Ala Leu Ala Leu Met Ser Arg Val Leu Asn Phe Pro Lys

165 170 175

Ser Ala Tyr Cys Gln Gln His Ser Ala Asp Gln Thr Cys Asp Phe Ala

180 185 190

Gln Ala Met Pro Ser Lys Leu Ser Ile Lys Asp Asp Gly Asn Lys Val

195 200 205

Ala Leu Asp Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu His Ala Gln Gly Met Pro Asn Ala Ala Trp Gly Lys Ile

225 230 235 240

His Ser Glu Gln Asp Trp Asn Ala Leu Leu Ala Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ser Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Ser Ala Ile Asn Ser Gln Thr Gly Thr

275 280 285

Arg Glu Leu Pro Glu Leu Ser Ala Asp Asn Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Leu Ser

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Trp Ser Asp Lys Ala Gly Lys Lys Tyr Val Ser Val

340 345 350

Gln Met Met Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Asp Glu Pro Ala Gly Ser Val Ala Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Gln Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Ala Lys Gln Asn Glu Leu Ala Glu Cys Gln

405 410

<210> 44

<211> 418

<212> PRT

<213> Rouxiella badensis

<400> 44

Asp Ala Pro Ala Thr Gln Asn Leu Gln Leu Gln Gln Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Gln Thr Lys Lys Met Glu

20 25 30

Gln Val Ala Ala Lys Asp Trp Pro Val Trp Pro Val Lys Phe Gly Tyr

35 40 45

Leu Thr Pro Arg Gly Glu His Leu Val Thr Leu Met Gly Gly Tyr Tyr

50 55 60

Gly Glu Tyr Phe Arg Lys Glu Gly Leu Ile Ser Asp Lys Ser Cys Pro

65 70 75 80

Ala Asn Gly Ala Ile Phe Gly Trp Gly Asp Val Asp Gln Arg Thr Arg

85 90 95

Leu Thr Thr Gln Ala Leu Leu Asn Gly Ile Ala Pro Gly Cys His Leu

100 105 110

Asp Ala His Phe Gln Asn Asp Leu Lys Lys Ala Asp Pro Leu Phe His

115 120 125

Ala Leu Lys Ala Lys Val Cys Lys Leu Asp Glu Lys Thr Ala Gln Gln

130 135 140

Ala Ile Glu Gln Gln Ala Gly Gly Ser Leu Ser Ala Leu Asp Lys Thr

145 150 155 160

Tyr Ala Pro Gln Leu Gln Leu Met Ser Asn Val Leu Asp Tyr Pro His

165 170 175

Ser Ala Tyr Cys Gln Lys Met Gln Lys Lys Gly Gln Gln Cys Glu Leu

180 185 190

Gly Ile Asn Met Pro Ser Ser Val Lys Val Lys Val Lys Val Lys Glu

195 200 205

Asn Gly Thr Asp Ala Ser Leu Lys Gly Ala Ile Gly Leu Ser Ser Thr

210 215 220

Leu Ala Glu Ile Phe Leu Leu Gln Asp Ala Gln Gly Met Thr Asp Pro

225 230 235 240

Ala Trp Gly Asn Ile Lys Asp Gln Lys Thr Trp Gln Gly Leu Met Ala

245 250 255

Leu His Asn Leu Gln Phe Ser Leu Met Ser Gly Thr Pro Tyr Leu Ala

260 265 270

Lys Ser Asn Gly Thr Pro Ile Leu Gln Ala Ile Asp Ser Ala Leu Gly

275 280 285

Ala Pro Glu Lys Pro Ala Ser Gly Tyr Thr Leu Pro Thr Gly Asn Lys

290 295 300

Leu Leu Ile Leu Gly Gly His Asp Thr Asn Ile Glu Asn Val Ala Gly

305 310 315 320

Ala Leu Gly Leu Asn Trp Ser Leu Asp Gly Gln Pro Asp Asn Thr Pro

325 330 335

Pro Ala Gly Ala Leu Val Phe Glu Arg Trp Gln Ser Arg Thr Ser His

340 345 350

Gln Gln Tyr Ile Ser Leu Lys Met Val Tyr Gln Thr Arg Asp Gln Met

355 360 365

Arg Ser Gln Gln Val Leu Thr Leu Asn Asn Pro Pro Ser Ser Ile Ala

370 375 380

Ile Thr Leu Pro Gly Cys Glu Asn Ile Gly Pro Asn Lys Leu Cys Ala

385 390 395 400

Ile Lys Thr Phe His Gln Val Met Thr Lys Ala Gln Leu Pro Gln Cys

405 410 415

Lys Ile

<210> 45

<211> 414

<212> PRT

<213> Unknown sequence

<220>

<223> Serratia sp. WP 009636981.1

<400> 45

Val Ile Pro Ala Pro Gln Asp Leu Gln Leu Gln Gln Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Lys Glu Met Lys

20 25 30

Asp Leu Ala Gly Gln Asp Trp Pro Lys Trp Pro Val Lys Pro Gly Tyr

35 40 45

Leu Thr Pro Arg Gly Gln Gln Leu Val Ser Leu Met Gly Thr Tyr Tyr

50 55 60

Gly Asp Tyr Phe Lys Lys Glu Gly Leu Leu Ser Ser Glu Gln Cys Pro

65 70 75 80

Gly Asp Ser Glu Val Phe Gly Trp Gly Asp Thr Asp Gln Arg Thr Arg

85 90 95

Leu Thr Thr Gln Thr Leu Leu Ser Ala Ile Ala Pro His Cys His Val

100 105 110

Met Ala Lys Asn Gln Ala Asp Leu Lys Lys Pro Asp Pro Val Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Thr Leu Asp Lys Ala Thr Ala Ile Lys

130 135 140

Ala Ile Asp Lys Glu Ala Gly Gly Ser Leu Asp Ala Leu Asp Gln Thr

145 150 155 160

Tyr Ala Pro Gln Leu Lys Leu Met Ser Gln Val Leu Asn Tyr Pro Gln

165 170 175

Ser Ser Tyr Cys Gln Gln Met Lys Gln Thr Gly Gln Thr Cys Ser Ala

180 185 190

Val Ile Asp Ile Pro Ser Ser Ile Lys Met Lys Lys Lys Gly Thr Glu

195 200 205

Ala Thr Leu Glu Gly Gly Ile Gly Met Ser Ser Thr Phe Ala Glu Asn

210 215 220

Phe Leu Leu Glu Asp Ala Gln Gly Met Gln Asp Val Ala Trp Gly Arg

225 230 235 240

Ile Lys Asp Gln Lys Thr Trp Gln Ala Leu Leu Glu Leu His Asn Leu

245 250 255

Gln Phe Arg Leu Met Ser Gly Thr Pro Tyr Ile Ala Lys Ser Asn Gly

260 265 270

Thr Pro Val Leu Gln Val Ile Asp Asn Ala Phe Gly Ala Ala Ala Pro

275 280 285

Ala Ser Ser Gly Phe Ser Leu Pro Ser Gly Asn Lys Val Leu Ile Leu

290 295 300

Gly Gly His Asp Thr Asn Ile Glu Asn Val Ala Gly Ala Leu Gly Leu

305 310 315 320

Asp Trp Thr Leu Thr Asp Gln Pro Asp His Thr Pro Pro Ala Gly Ala

325 330 335

Leu Met Phe Glu Arg Trp Gln Asp Lys Thr Thr His Gln Gln Tyr Ile

340 345 350

Ser Leu Arg Met Val Tyr Gln Thr Gln Asp Gln Met Arg Thr Gln His

355 360 365

Lys Leu Thr Leu Lys His Pro Pro Met Thr Val Ala Leu Ser Ile Pro

370 375 380

Gly Cys Glu Asn Ile Gly Asp Asn Lys Leu Cys Ala Ile Gly Thr Phe

385 390 395 400

His Gln Val Ile Glu Lys Ala Gln Leu Pro Gln Cys Lys Ile

405 410

<210> 46

<211> 412

<212> PRT

<213> Yersinia aldovae

<400> 46

Gln Pro Ala Gly Tyr Thr Leu Glu Arg Val Val Ile Leu Ser Arg His

1 5 10 15

Gly Val Arg Ser Pro Thr Lys Gln Thr Gln Leu Met Asn Asp Val Thr

20 25 30

Pro Asp Lys Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr Leu Thr Pro

35 40 45

Arg Gly Ala Gln Leu Val Thr Leu Met Gly Gln Phe Tyr Gly Asp Tyr

50 55 60

Phe Arg Ser Lys Gly Leu Leu Leu Ala Gly Cys Pro Ala Glu Gly Val

65 70 75 80

Ile Tyr Ala Gln Ala Asp Ile Asp Gln Arg Thr Arg Leu Thr Gly Gln

85 90 95

Ala Phe Leu Asp Gly Val Ala Pro Asp Cys Gly Leu Lys Val His Tyr

100 105 110

Gln Ala Asp Leu Lys Lys Thr Asp Pro Leu Phe His Pro Val Glu Ala

115 120 125

Gly Val Cys Lys Leu Asp Ala Val Gln Thr Gln Lys Ala Val Glu Glu

130 135 140

His Leu Gly Gly Pro Leu Ser Ser Leu Gly Glu Arg Tyr Thr Lys Pro

145 150 155 160

Phe Ala Gln Met Gly Glu Val Leu Asn Phe Ala Lys Ser Pro Tyr Cys

165 170 175

Lys Thr Arg Gln Gln Asn Asp Lys Thr Cys Asp Phe Ala His Phe Ala

180 185 190

Ala Asn Glu Ile Lys Val Asn Lys Glu Gly Ser Lys Val Ser Leu Asn

195 200 205

Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe Leu Leu Gln

210 215 220

Asn Ala Gln Asn Met Pro Asn Val Ala Trp Asn Arg Leu Ser Gly Thr

225 230 235 240

Glu Asn Trp Ala Ser Leu Leu Ser Leu His Asn Val Gln Phe Asp Leu

245 250 255

Met Ala Lys Thr Pro Tyr Ile Ala Arg His Lys Gly Thr Pro Leu Leu

260 265 270

Gln Gln Ile Asp Ala Ala Leu Thr Leu Gln Pro Asp Ala Leu Gly Gln

275 280 285

Thr Leu Pro Leu Ser Pro Gln Ser Arg Val Leu Phe Ile Gly Gly His

290 295 300

Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Ala Ser Trp Gln

305 310 315 320

Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly Leu Val Phe

325 330 335

Glu Leu Trp Gln Asn Pro Asp Asn His Gln Arg Tyr Val Ala Val Lys

340 345 350

Met Phe Tyr Gln Thr Met Asp Gln Leu Arg Lys Ala Glu Met Leu Asp

355 360 365

Leu Lys Asn Asn Pro Ala Gly Met Ile Ser Val Ala Val Glu Gly Cys

370 375 380

Glu Asn Ser Gly Asp Asp Lys Leu Cys Gln Leu Asp Thr Phe Gln Lys

385 390 395 400

Lys Val Ala Gln Val Ile Glu Pro Ala Cys His Ile

405 410

<210> 47

<211> 415

<212> PRT

<213> Yersinia frederiksenii

<400> 47

Val Val Ala Pro Pro Thr Gly Tyr Thr Leu Glu Arg Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln Leu Met Asn

20 25 30

Asp Val Thr Pro Asp Lys Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr

35 40 45

Leu Thr Pro Arg Gly Ala Glu Leu Val Thr Leu Met Gly Gly Phe Tyr

50 55 60

Gly Asp Tyr Phe Arg His Gln Gly Leu Leu Pro Met Gly Cys Pro Ala

65 70 75 80

Asp Gly Ala Ile Tyr Ala Gln Ala Asp Val Asp Gln Arg Thr Arg Leu

85 90 95

Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys Gly Leu Thr

100 105 110

Val His Tyr Gln Ala Asp Leu Lys Lys Ile Asp Pro Leu Phe His Pro

115 120 125

Val Glu Ala Gly Val Cys Gln Leu Asp Ser Thr Gln Thr His Lys Ala

130 135 140

Val Glu Glu Arg Leu Gly Gly Pro Leu Asn Thr Leu Ser Gln Arg Tyr

145 150 155 160

Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe Ser Thr Ser

165 170 175

Pro Tyr Cys Gln Ser Leu Gln Gln Thr Gly Lys Thr Cys Asp Phe Ala

180 185 190

Thr Phe Ala Ala Asn Glu Ile Thr Val Asn Lys Ala Gly Thr Lys Val

195 200 205

Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe

210 215 220

Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp His Arg Leu

225 230 235 240

Asn Gly Ala Glu Asn Trp Ala Ser Leu Leu Ser Leu His Asn Val Gln

245 250 255

Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His Lys Gly Thr

260 265 270

Pro Leu Leu Gln Gln Ile Glu Thr Ala Leu Leu Leu Gln Arg Asp Ala

275 280 285

Gln Gly Gln Lys Leu Pro Leu Ser Pro Gln Thr Lys Val Leu Phe Leu

290 295 300

Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Ala

305 310 315 320

Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly

325 330 335

Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln Arg Tyr Val

340 345 350

Ala Val Lys Met Phe Tyr Gln Thr Met Asp Gln Leu Arg Arg Ala Glu

355 360 365

Lys Leu Asp Leu Lys Asn Asn Pro Ala Gly Ile Val Pro Ile Thr Val

370 375 380

Glu Glu Cys Glu Asn Thr Gly Asp Asn Lys Leu Cys Gln Leu Glu Thr

385 390 395 400

Phe Gln Lys Lys Val Ala Lys Met Ile Glu Pro Ala Cys His Ile

405 410 415

<210> 48

<211> 415

<212> PRT

<213> Yersinia kristensenii

<400> 48

Leu Ala Ala Gln Ser Thr Gly Tyr Thr Leu Glu Arg Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln Leu Met Asn

20 25 30

Asp Val Thr Pro Asp Lys Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr

35 40 45

Leu Thr Pro Arg Gly Ala Gly Leu Val Thr Leu Met Gly Gly Phe Tyr

50 55 60

Gly Asp Tyr Phe Arg Ser Tyr Gly Leu Leu Pro Ala Gly Cys Pro Ala

65 70 75 80

Asp Glu Ser Ile Tyr Val Gln Ala Asp Val Asp Gln Arg Thr Arg Leu

85 90 95

Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Asp Cys Gly Leu Lys

100 105 110

Val His Tyr Gln Ala Asp Leu Lys Lys Ile Asp Pro Leu Phe His Thr

115 120 125

Val Glu Ala Gly Val Cys Lys Leu Asp Pro Glu Lys Thr His Gln Ala

130 135 140

Val Glu Lys Arg Leu Gly Gly Pro Leu Asn Glu Leu Ser Gln Arg Tyr

145 150 155 160

Ala Lys Pro Phe Ala Leu Met Gly Glu Val Leu Asn Phe Ser Ala Ser

165 170 175

Pro Tyr Cys Asn Ser Leu Gln Gln Lys Gly Lys Thr Cys Asp Phe Ala

180 185 190

Thr Phe Ala Ala Asn Glu Ile Glu Val Asn Lys Glu Gly Thr Lys Val

195 200 205

Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe

210 215 220

Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp Asn Arg Leu

225 230 235 240

Ser Gly Glu Glu Asn Trp Ile Ser Leu Leu Ser Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His Lys Gly Thr

260 265 270

Pro Leu Leu Gln Gln Ile Asp Thr Ala Leu Val Leu Gln Arg Asp Ala

275 280 285

Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Leu Leu Phe Leu

290 295 300

Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Ala

305 310 315 320

Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly

325 330 335

Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln Arg Tyr Val

340 345 350

Ala Val Lys Met Phe Tyr Gln Thr Met Glu Gln Leu Arg Asn Ala Asp

355 360 365

Lys Leu Asp Leu Lys Asn Asn Pro Ala Arg Ile Val Pro Ile Ala Ile

370 375 380

Glu Gly Cys Glu Asn Glu Gly Asp Asn Lys Leu Cys Gln Leu Glu Thr

385 390 395 400

Phe Gln Lys Lys Val Ala Gln Val Ile Glu Pro Thr Cys His Ile

405 410 415

<210> 49

<211> 415

<212> PRT

<213> Yersinia mollaretii

<400> 49

Val Ala Ala Pro Ala Ala Gly Tyr Thr Leu Glu Arg Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln Leu Met Asn

20 25 30

Asp Val Thr Pro Asp Lys Trp Pro Gln Trp Pro Val Gln Ala Gly Tyr

35 40 45

Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Met Gly Gly Phe Tyr

50 55 60

Gly Asp Tyr Phe Arg Ser Gln Gly Leu Leu Pro Thr Gly Cys Pro Ala

65 70 75 80

Asp Gly Thr Leu Tyr Ala Gln Ala Asp Ile Asp Gln Arg Thr Arg Leu

85 90 95

Thr Gly Gln Ala Phe Leu Asp Gly Ile Ala Pro Gly Cys Asp Leu Lys

100 105 110

Val His Tyr Gln Ala Asp Leu Lys Lys Val Asp Pro Leu Phe His Pro

115 120 125

Val Glu Ala Gly Val Cys Gln Leu Asp Ser Ala Gln Thr His Gln Ala

130 135 140

Ile Glu Ala Arg Leu Gly Ala Pro Leu Ser Glu Leu Ser Gln Arg Tyr

145 150 155 160

Ala Lys Pro Phe Ala Gln Met Gly Glu Ile Leu Asn Phe Ala Ala Ser

165 170 175

Pro Tyr Cys Lys Ser Leu Gln Gln Gln Gly Lys Ser Cys Asp Phe Ala

180 185 190

Thr Phe Ala Ala Asn Glu Val Lys Val Asn Gln Gln Gly Thr Lys Val

195 200 205

Ser Leu Ser Gly Pro Leu Ala Leu Ser Ser Thr Leu Gly Glu Ile Phe

210 215 220

Leu Leu Gln Asn Ser Gln Gly Met Pro Asp Val Ala Trp His Arg Leu

225 230 235 240

Ser Gly Ala Glu Asn Trp Val Ser Leu Leu Ser Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His Lys Gly Thr

260 265 270

Pro Leu Leu Gln Gln Ile Met Thr Ala Leu Val Leu Gln Arg Lys Gly

275 280 285

Gln Gly Gln Thr Leu Pro Leu Ser Glu Gln Thr Lys Leu Leu Phe Leu

290 295 300

Gly Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Ala

305 310 315 320

Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly

325 330 335

Leu Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln Gln Tyr Val

340 345 350

Ala Val Lys Met Phe Tyr Gln Thr Met Asp Gln Leu Arg Asn Ser Glu

355 360 365

Lys Leu Asp Leu Lys Ser His Pro Ala Gly Ile Val Pro Ile Glu Ile

370 375 380

Glu Ser Cys Glu Asn Ile Gly Thr Asp Lys Leu Cys Gln Leu Asp Thr

385 390 395 400

Phe Gln Lys Arg Val Ala Gln Val Ile Glu Pro Ala Cys His Ile

405 410 415

<210> 50

<211> 415

<212> PRT

<213> Yersinia rohdei

<400> 50

Val Ile Thr Ala Pro Ala Gly Tyr Thr Leu Glu Arg Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ser Pro Thr Lys Gln Thr Gln Leu Met Asn

20 25 30

Glu Val Thr Pro Asp Lys Trp Pro Gln Trp Pro Val Lys Ala Gly Tyr

35 40 45

Leu Thr Pro Arg Gly Ala Gln Leu Val Thr Leu Leu Gly Ala Phe Tyr

50 55 60

Gly Glu Tyr Phe Arg Ser Gln Gly Leu Leu Pro Ala Gly Cys Pro Pro

65 70 75 80

Glu Gly Thr Val Tyr Ala Gln Ala Asp Ile Asp Gln Arg Thr Arg Leu

85 90 95

Thr Gly Gln Ala Phe Leu Asp Gly Val Ala Pro Gly Cys Gly Leu Glu

100 105 110

Val His Tyr Gln Ala Asp Leu Lys Lys Thr Asp Pro Leu Phe His Pro

115 120 125

Val Glu Ala Gly Val Cys Lys Val Asp Leu Ala Gln Thr Arg Gln Ala

130 135 140

Val Glu Gln Arg Leu Gly Gly Pro Leu Thr Thr Leu Ser Gln Arg Tyr

145 150 155 160

Ala Lys Pro Phe Ala Gln Met Gly Glu Val Leu Asn Phe Ala Glu Ser

165 170 175

Pro Phe Cys Lys Ser Leu Gln Gln Lys Gly Lys Thr Cys Asp Phe Ala

180 185 190

Thr Phe Ala Ala Asn Glu Ile Asp Val Asn Lys Asp Gly Thr Lys Ile

195 200 205

Ser Leu Thr Gly Pro Leu Ala Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Gln Asn Ser Gln Ala Met Pro Asp Val Ala Trp His Arg Leu

225 230 235 240

Ser Gly Ala Glu Asn Trp Val Ser Leu Leu Ser Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ala Lys Thr Pro Tyr Ile Ala Arg His Lys Gly Thr

260 265 270

Pro Leu Leu Gln Gln Ile Asn Thr Ala Leu Val Leu Gln Arg Asp Ala

275 280 285

Gln Gly Gln Thr Leu Pro Leu Ser Pro Gln Thr Lys Val Leu Phe Leu

290 295 300

Gly Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Ala

305 310 315 320

Asn Trp Gln Leu Pro Gln Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly

325 330 335

Leu Val Phe Glu Leu Trp Gln His Pro Asp Asn His Gln Arg Tyr Val

340 345 350

Ala Val Lys Met Phe Tyr Gln Thr Met Asp Gln Leu Arg Asn Val Glu

355 360 365

Lys Leu Asn Leu Thr Thr Asn Pro Ala Gly Ile Ile Pro Ile Ala Val

370 375 380

Glu Gly Cys Glu Asn Met Gly Asp Asp Lys Leu Cys Gln Leu Glu Thr

385 390 395 400

Phe Glu Lys Lys Ile Ala Gln Val Ile Glu Pro Ala Cys His Ile

405 410 415

<210> 51

<211> 410

<212> PRT

<213> Escherichia coli

<400> 51

Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser Arg

1 5 10 15

His Gly Val Arg Ala Pro Thr Lys Phe Thr Gln Leu Met Gln Asp Val

20 25 30

Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu Thr

35 40 45

Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg Gln

50 55 60

Arg Leu Val Ala Asp Glu Leu Leu Pro Lys Cys Gly Cys Pro Gln Ser

65 70 75 80

Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr

85 90 95

Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val

100 105 110

His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu

115 120 125

Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Thr Arg Ala Ile

130 135 140

Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Tyr Gln

145 150 155 160

Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn

165 170 175

Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr Gln

180 185 190

Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr

195 200 205

Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln

210 215 220

Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser

225 230 235 240

His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu

245 250 255

Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu

260 265 270

Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala

275 280 285

Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp

290 295 300

Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu

305 310 315 320

Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu

325 330 335

Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu

340 345 350

Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser Leu

355 360 365

Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu

370 375 380

Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val

385 390 395 400

Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu

405 410

<210> 52

<211> 413

<212> PRT

<213> Unknown sequence

<220>

<223> US8101391-0002

<400> 52

Asn Asp Thr Pro Ala Ser Gly Tyr Gln Val Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asn Thr Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Glu Lys Phe Gln Gln Gln Gly Ile Leu Ser Gln Gly Ser Cys Pro

65 70 75 80

Thr Pro Asn Ser Ile Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Leu

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Gly Leu

100 105 110

Thr Ile His His Gln Gln Asn Leu Glu Lys Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Thr Cys Ser Met Asp Lys Thr Arg Leu Gln Gln

130 135 140

Ala Val Glu Lys Glu Ala Gln Thr Pro Ile Glu Asn Leu Asn Gln His

145 150 155 160

Tyr Ile Pro Ser Leu Ala Leu Met Asn Thr Thr Leu Asn Phe Ser Thr

165 170 175

Ser Ala Trp Cys Gln Lys His Ser Ala Asp Lys Ser Cys Asp Leu Ala

180 185 190

Gln Ser Met Pro Ser Lys Leu Ser Ile Lys Asp Asn Gly Asn Lys Val

195 200 205

Ala Leu Asp Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Gln Ala Ala Trp Gly Lys Ile

225 230 235 240

His Ser Glu Gln Asp Trp Ala Glu Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ala Arg Thr Pro Tyr Ile Ala Arg His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asp Pro Asn Ala Thr Ala

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ser Gly Met Leu Asn Met Arg

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Ile Phe Glu Arg Leu Ala Asp Lys Ala Gly Lys Gln Tyr Val Ser Val

340 345 350

Ser Met Val Tyr Gln Thr Leu Glu Gln Leu Arg Ala Gln Thr Pro Leu

355 360 365

Ser Leu Lys Glu Pro Ala Gly Ser Val Gln Leu Lys Ile Pro Gly Cys

370 375 380

Asn Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Pro Thr Phe Lys Arg

385 390 395 400

Val Val Ser Gln Ser Glu Glu Pro Gly Cys Gln Leu Gln

405 410

<210> 53

<211> 413

<212> PRT

<213> Unknown sequence

<220>

<223> US8101391-0004

<400> 53

Ser Asp Thr Pro Ala Ser Gly Tyr Gln Ile Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asn Ser Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Lys Phe Gln Gln Lys Gly Ile Leu Ser Gln Gly Ser Cys Pro

65 70 75 80

Thr Pro Asn Ser Ile Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Leu

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Gly Leu

100 105 110

Thr Ile His His Gln Gln Asn Leu Glu Lys Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Thr Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Glu Ala Gln Met Pro Ile Glu Asn Leu Asn Gln His

145 150 155 160

Tyr Ile Pro Ser Leu Ala Leu Met Asn Thr Thr Leu Asn Phe Ser Thr

165 170 175

Ser Ala Trp Cys Gln Lys His Ser Ala Asp Lys Ser Cys Asp Leu Ala

180 185 190

Gln Ser Met Pro Ser Lys Leu Ser Ile Lys Asp Asn Gly Asn Lys Val

195 200 205

Ala Leu Asp Gly Ala Ile Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Gln Ala Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Ala Ser Leu Leu Lys Leu His Asn Thr Gln

245 250 255

Phe Asp Leu Met Ala Arg Thr Pro Tyr Ile Ala Ala His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Ser Asn Ala Leu Glu Pro Lys Ala Asp Val

275 280 285

Ser Lys Leu Pro Asp Ile Ser Ser Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Asn Met Arg

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Leu Ala Asp Lys Ser Gly Lys Gln Tyr Ile Ser Val

340 345 350

Ser Met Val Tyr Gln Thr Leu Glu Gln Leu Arg Ala Gln Thr Pro Leu

355 360 365

Ser Leu Asn Glu Pro Ala Gly Ser Val Gln Leu Lys Ile Pro Gly Cys

370 375 380

Asn Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Ser Thr Phe Thr Arg

385 390 395 400

Val Val Ser Gln Ser Val Glu Pro Gly Cys Gln Leu Pro

405 410

<210> 54

<211> 413

<212> PRT

<213> Pyrococcus yayanosi

<400> 54

Asn Asp Thr Pro Ala Ser Gly Tyr Gln Val Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asn Thr Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Lys Phe Gln Gln Gln Gly Ile Leu Ser Gln Gly Ser Cys Pro

65 70 75 80

Thr Pro Asn Ser Ile Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Leu

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys His Leu

100 105 110

Thr Ile His His Gln Gln Asp Ile Lys Lys Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Thr Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Glu Ala Gln Thr Pro Ile Asp Asn Leu Asn Gln His

145 150 155 160

Tyr Ile Pro Phe Leu Ala Leu Met Asn Thr Thr Leu Asn Phe Ser Lys

165 170 175

Ser Ala Trp Cys Gln Lys His Ser Ala Asp Lys Ser Cys Asp Leu Gly

180 185 190

Leu Ser Met Pro Ser Lys Leu Ser Ile Lys Asp Asn Gly Asn Lys Val

195 200 205

Ala Leu Asp Gly Ala Ile Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Gln Ala Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Ala Ser Leu Leu Lys Leu His Asn Val Gln

245 250 255

Phe Asp Leu Met Ala Arg Thr Pro Tyr Ile Ala Arg His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Asn Met Arg

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Leu Ala Asp Lys Ser Gly Lys Gln Tyr Val Ser Val

340 345 350

Ser Met Val Tyr Gln Thr Leu Glu Gln Leu Arg Ser Gln Thr Pro Leu

355 360 365

Ser Leu Asn Gln Pro Ala Gly Ser Val Gln Leu Lys Ile Pro Gly Cys

370 375 380

Asn Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Ser Thr Phe Thr Arg

385 390 395 400

Val Val Ser Gln Ser Val Glu Pro Gly Cys Gln Leu Gln

405 410

<210> 55

<211> 411

<212> PRT

<213> Obesumbacterium proteus

<400> 55

Ser Glu Thr Glu Pro Ser Gly Tyr Gln Leu Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asn Ala Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Lys Phe Gln Gln Leu Gly Ile Leu Ser Lys Gly Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys His Leu

100 105 110

Ser Ile His His Gln Gln Asp Ile Lys Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Val Cys Thr Met Glu Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Gln Gln Ala Gly Met Pro Ile Asp Gln Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Ala Leu Ala Leu Met Ser Ser Val Leu Asn Phe Pro Lys

165 170 175

Ser Thr Tyr Cys Gln Gln His Ser Ala Asp Gln Thr Cys Asp Leu Ala

180 185 190

Gln Ala Met Pro Ser Lys Leu Ser Ile Lys Asp Asn Gly Asn Lys Val

195 200 205

Ala Leu Asp Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Asp Ala Ala Trp Gly Lys Ile

225 230 235 240

His Ser Glu Gln Asp Trp Asn Ala Leu Leu Thr Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ser Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Ser Ala Ile Asn Ser Gln Pro Ser Ser

275 280 285

Arg Glu Leu Pro Glu Leu Ser Ala Asp Asn Lys Ile Leu Phe Pro Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Phe Gly Met Ser

305 310 315 320

Trp Ala Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Trp Ser Asp Lys Thr Gly Lys Lys Tyr Val Ser Val

340 345 350

Gln Met Met Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Asp Lys Pro Ala Gly Ser Val Ala Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Ala Lys Gln Asn Glu Leu Val Glu Cys Gln

405 410

<210> 56

<211> 413

<212> PRT

<213> Unknown sequence

<220>

<223> US8143046-0001

<400> 56

Asn Asp Thr Pro Ala Ser Gly Tyr Gln Val Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asn Thr Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Lys Phe Gln Gln Gln Gly Ile Leu Ser Gln Gly Ser Cys Pro

65 70 75 80

Thr Pro Asn Ser Ile Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Leu

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys His Leu

100 105 110

Thr Ile His His Gln Gln Asp Ile Lys Lys Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Thr Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Glu Ala Gln Thr Pro Ile Asp Asn Leu Asn Gln His

145 150 155 160

Tyr Ile Pro Phe Leu Ala Leu Met Asn Thr Thr Leu Asn Phe Ser Thr

165 170 175

Ser Ala Trp Cys Gln Lys His Ser Ala Asp Lys Ser Cys Asp Leu Gly

180 185 190

Leu Ser Met Pro Ser Lys Leu Ser Ile Lys Asp Asn Gly Asn Lys Val

195 200 205

Ala Leu Asp Gly Ala Ile Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Gln Ala Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Ala Ser Leu Leu Lys Leu His Asn Val Gln

245 250 255

Phe Asp Leu Met Ala Arg Thr Pro Tyr Ile Ala Arg His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Asn Met Arg

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Leu Ala Asp Lys Ser Gly Lys Gln Tyr Val Ser Val

340 345 350

Ser Met Val Tyr Gln Thr Leu Glu Gln Leu Arg Ser Gln Thr Pro Leu

355 360 365

Ser Leu Asn Gln Pro Ala Gly Ser Val Gln Leu Lys Ile Pro Gly Cys

370 375 380

Asn Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Ser Thr Phe Thr Arg

385 390 395 400

Val Val Ser Gln Ser Val Glu Pro Gly Cys Gln Leu Gln

405 410

<210> 57

<211> 413

<212> PRT

<213> Unknown sequence

<220>

<223> US8143046-0003US8143046-0003

<400> 57

Asn Asp Thr Pro Ala Ser Gly Tyr Gln Val Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro Asn Thr Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Lys Phe Gln Gln Gln Gly Ile Leu Ser Gln Gly Ser Cys Pro

65 70 75 80

Thr Pro Asn Ser Ile Tyr Val Trp Thr Asp Val Ala Gln Arg Thr Leu

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Gly Leu

100 105 110

Thr Ile His His Gln Gln Asn Leu Glu Lys Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Glu Ala Gln Thr Pro Ile Asp Asn Leu Asn Gln His

145 150 155 160

Tyr Ile Pro Ser Leu Ala Leu Met Asn Thr Thr Leu Asn Phe Ser Lys

165 170 175

Ser Pro Trp Cys Gln Lys His Ser Ala Asp Lys Ser Cys Asp Leu Gly

180 185 190

Leu Ser Met Pro Ser Lys Leu Ser Ile Lys Asp Asn Gly Asn Glu Val

195 200 205

Ser Leu Asp Gly Ala Ile Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Gln Ala Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Ala Leu Leu Leu Lys Leu His Asn Val Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Arg His Lys Gly Thr

260 265 270

Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Asn Met Arg

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Leu Ala Asp Lys Ser Gly Lys Gln Tyr Val Ser Val

340 345 350

Ser Met Val Tyr Gln Thr Leu Glu Gln Leu Arg Ser Gln Thr Pro Leu

355 360 365

Ser Leu Asn Gln Pro Ala Gly Ser Val Gln Leu Lys Ile Pro Gly Cys

370 375 380

Asn Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Ser Thr Phe Thr Arg

385 390 395 400

Val Val Ser Gln Ser Val Glu Pro Gly Cys Gln Leu Gln

405 410

<210> 58

<211> 411

<212> PRT

<213> Citrobacter braakii

<400> 58

Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val Val Ile Val Ser Arg

1 5 10 15

His Gly Val Arg Ala Pro Thr Lys Phe Thr Pro Ile Met Lys Asn Val

20 25 30

Thr Pro Asp Gln Trp Pro Gln Trp Asp Val Pro Leu Gly Trp Leu Thr

35 40 45

Pro Arg Gly Gly Glu Leu Val Ser Glu Leu Gly Gln Tyr Gln Arg Leu

50 55 60

Trp Phe Thr Ser Lys Gly Leu Leu Asn Asn Gln Thr Cys Pro Ser Pro

65 70 75 80

Gly Gln Val Ala Val Ile Ala Asp Thr Asp Gln Arg Thr Arg Lys Thr

85 90 95

Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Lys Cys Gln Ile Gln Val

100 105 110

His Tyr Gln Lys Asp Glu Glu Lys Asn Asp Pro Leu Phe Asn Pro Val

115 120 125

Lys Met Gly Lys Cys Ser Phe Asn Thr Leu Gln Val Lys Asn Ala Ile

130 135 140

Leu Glu Arg Ala Gly Gly Asn Ile Glu Leu Tyr Thr Gln Arg Tyr Gln

145 150 155 160

Ser Ser Phe Arg Thr Leu Glu Asn Val Leu Asn Phe Ser Gln Ser Glu

165 170 175

Thr Cys Lys Thr Thr Glu Lys Ser Thr Lys Cys Thr Leu Pro Glu Ala

180 185 190

Leu Pro Ser Glu Leu Lys Val Thr Pro Asp Asn Val Ser Leu Pro Gly

195 200 205

Ala Trp Ser Leu Ser Ser Thr Leu Thr Glu Ile Phe Leu Leu Gln Glu

210 215 220

Ala Gln Gly Met Pro Gln Val Ala Trp Gly Arg Ile Thr Gly Glu Lys

225 230 235 240

Glu Trp Arg Asp Leu Leu Ser Leu His Asn Ala Gln Phe Asp Leu Leu

245 250 255

Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu Asp

260 265 270

Met Ile Asp Thr Ala Leu Leu Thr Asn Gly Thr Thr Glu Asn Arg Tyr

275 280 285

Gly Ile Lys Leu Pro Val Ser Leu Leu Phe Ile Ala Gly His Asp Thr

290 295 300

Asn Leu Ala Asn Leu Ser Gly Ala Leu Asp Leu Asn Trp Ser Leu Pro

305 310 315 320

Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu Lys

325 330 335

Trp Lys Arg Thr Ser Asp Asn Thr Asp Trp Val Gln Val Ser Phe Val

340 345 350

Tyr Gln Thr Leu Arg Asp Met Arg Asp Ile Gln Pro Leu Ser Leu Glu

355 360 365

Lys Pro Ala Gly Lys Val Asp Leu Lys Leu Ile Ala Cys Glu Glu Lys

370 375 380

Asn Ser Gln Gly Met Cys Ser Leu Lys Ser Phe Ser Arg Leu Ile Lys

385 390 395 400

Glu Ile Arg Val Pro Glu Cys Ala Val Thr Glu

405 410

<210> 59

<211> 413

<212> PRT

<213> Unknown sequence

<220>

<223> US8557555-0013

<400> 59

Ser Asp Thr Ala Pro Ala Gly Phe Gln Leu Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asn Val Thr Pro His Gln Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Glu Arg Phe Gln Gln Gln Gly Leu Leu Pro Lys Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Ala Val Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Asp Leu

100 105 110

Ala Ile His His Gln Gln Asn Ile Gln Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Ser Gln Ala His Ala

130 135 140

Ala Val Glu Lys Gln Ala Gly Thr Pro Ile Glu Thr Leu Asn Gln Arg

145 150 155 160

Tyr Gln Ala Ser Leu Ala Leu Met Ser Ser Val Leu Asp Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Gln His Asn Ile Gly Lys Leu Cys Asp Phe Ser

180 185 190

Gln Ala Met Pro Ser Arg Leu Ala Ile Asn Asp Asp Gly Asn Lys Val

195 200 205

Ala Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu His Ala Gln Gly Met Pro Lys Val Ala Trp Gly Asn Ile

225 230 235 240

His Thr Glu Gln Gln Trp Asp Ser Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ser Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Ala His Ala Leu Gly Ser Asn Ile Ala Ser

275 280 285

Arg Pro Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ser Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Trp Val Asp Asn Ala Gly Lys Pro Tyr Val Ser Val

340 345 350

Asn Met Val Tyr Gln Thr Leu Ala Gln Leu His Asp Gln Thr Pro Leu

355 360 365

Thr Leu Gln His Pro Ala Gly Ser Val Arg Leu Asn Ile Pro Gly Cys

370 375 380

Ser Asp Gln Thr Pro Asp Gly Tyr Cys Pro Leu Ser Thr Phe Ser Arg

385 390 395 400

Leu Val Asn His Ser Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 60

<211> 414

<212> PRT

<213> Unknown sequence

<220>

<223> US8557555-0024

<400> 60

Ser Asp Thr Ala Pro Ala Gly Phe Gln Leu Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro His Gln Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Glu Arg Phe Gln Gln Gln Gly Leu Leu Pro Lys Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Ala Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Asp Leu

100 105 110

Ala Ile His His Gln Gln Asn Ile Thr Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asn Lys Ser Gln Thr Tyr Glu

130 135 140

Ala Val Glu Lys Gln Ala Gly Gly Pro Ile Glu Thr Leu Asn Gln Arg

145 150 155 160

Tyr Gln Ala Glu Leu Ala Leu Met Ser Ser Val Leu Asp Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Gln His Asn Ile Gly Lys Leu Cys Asp Phe Ser

180 185 190

Gln Ala Met Pro Ser Arg Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Gly Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Asn Glu Ser Gln Trp Lys Ser Leu Leu Asn Leu His Asn Ala His

245 250 255

Phe Asn Leu Met His Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Ala Ile Ser Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asn

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly Leu

325 330 335

Val Phe Glu Leu Trp Gln Asn Pro Asp Asn His Gln Gln Tyr Val Ala

340 345 350

Val Lys Met Ile Tyr Gln Thr Met Asp Gln Leu Arg Asn Ser Glu Lys

355 360 365

Leu Asp Leu Lys Ser Asn Pro Ala Gly Ile Val Pro Ile Glu Ile Glu

370 375 380

Gly Cys Glu Asn Ile Gly Thr Asp Lys Leu Cys Gln Leu Asp Thr Phe

385 390 395 400

Gln Lys Arg Val Ala Gln Val Ile Glu Pro Ala Cys Gln Ile

405 410

<210> 61

<211> 411

<212> PRT

<213> Escherichia coli (E. coli) K12

<400> 61

Met Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val Val Ile Val Ser

1 5 10 15

Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp

20 25 30

Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Glu Leu

35 40 45

Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Trp Arg

50 55 60

Gln Arg Leu Val Ala Asp Gly Leu Leu Pro Lys Glu Gly Cys Pro Gln

65 70 75 80

Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys

85 90 95

Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr

100 105 110

Val His His Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro

115 120 125

Leu Lys Thr Gly Val Cys Gln Leu Asp Val Ala Asn Val Arg Arg Ala

130 135 140

Ile Leu Arg Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Arg His Tyr

145 150 155 160

Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser

165 170 175

Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser Cys Ser Leu Thr

180 185 190

Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asp Val Ser Leu

195 200 205

Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu

210 215 220

Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp

225 230 235 240

Ser His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Val Phe Asp

245 250 255

Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu

260 265 270

Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln

275 280 285

Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His

290 295 300

Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr

305 310 315 320

Leu Pro Gly Gln Pro Asp Asn Tyr Pro Pro Gly Gly Glu Leu Val Phe

325 330 335

Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser

340 345 350

Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys Thr Pro Leu Ser

355 360 365

Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu

370 375 380

Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile

385 390 395 400

Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu

405 410

<210> 62

<211> 413

<212> PRT

<213> Unknown sequence

<220>

<223> WO2010034835-0001

<400> 62

Ser Asp Thr Ala Pro Ala Gly Phe Gln Leu Glu Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Thr Met Arg

20 25 30

Asp Val Thr Pro His Gln Trp Pro Glu Trp Pro Val Lys Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Glu Arg Phe Gln Gln Gln Gly Leu Leu Pro Lys Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Ala Val Tyr Val Trp Ala Asp Val Asp Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Gln Cys Asp Leu

100 105 110

Ala Ile His His Gln Gln Asn Thr Gln Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Ser Gln Val His Ala

130 135 140

Ala Val Glu Lys Gln Ala Gly Thr Pro Ile Glu Thr Leu Asn Gln Arg

145 150 155 160

Tyr Gln Ala Ser Leu Ala Leu Met Ser Ser Val Leu Asp Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Gln His Asn Ile Gly Lys Leu Cys Asp Phe Ser

180 185 190

Gln Ala Met Pro Ser Arg Leu Ala Ile Asn Asp Asp Gly Asn Lys Val

195 200 205

Ala Leu Glu Gly Ala Val Gly Leu Ala Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu His Ala Gln Gly Met Pro Lys Val Ala Trp Gly Asn Ile

225 230 235 240

His Thr Glu Gln Gln Trp Asn Ser Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Ser Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Ala His Ala Leu Gly Ser Asn Ile Thr Ser

275 280 285

Arg Pro Leu Pro Asp Ile Ser Pro Asp Asn Lys Ile Leu Phe Ile Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ser Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Arg Trp Val Asp Asn Ala Gly Lys Pro Tyr Val Ser Val

340 345 350

Asn Met Val Tyr Gln Thr Leu Ala Gln Leu His Asp Gln Ala Pro Leu

355 360 365

Thr Leu Gln His Pro Ala Gly Ser Val Arg Leu Asn Ile Pro Gly Cys

370 375 380

Ser Asp Gln Thr Pro Asp Gly Tyr Cys Pro Leu Ser Thr Phe Ser Arg

385 390 395 400

Leu Val Ser His Ser Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 63

<211> 20

<212> PRT

<213> Trichoderma reesei

<400> 63

Met Gln Thr Phe Gly Ala Phe Leu Val Ser Phe Leu Ala Ala Ser Gly

1 5 10 15

Leu Ala Ala Ala

20

<210> 64

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 64

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Ala

275 280 285

Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 65

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 65

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Ile Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Val Asp Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Met Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Arg Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Ala Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 66

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 66

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Val Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Gln His Ser Gly Asp Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala His

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 67

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 67

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 68

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 68

Ser Glu Thr Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 69

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 69

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 70

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 70

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Leu Trp Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 71

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 71

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Asp Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 72

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 72

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Ala

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 73

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 73

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Ala Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 74

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 74

Ser Glu Ala Ala Pro Ala Gly Tyr Gln Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 75

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 75

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 76

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 76

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Val Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 77

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 77

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Ile Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 78

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 78

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Ser Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 79

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 79

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 80

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 80

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 81

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 81

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Ala Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 82

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 82

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Lys Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 83

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 83

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Gln Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 84

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 84

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Arg Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 85

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 85

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Lys Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 86

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 86

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Arg Cys Pro

65 70 75 80

Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys Asp Phe Ala

180 185 190

Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Gln

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Gln Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn Thr Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Thr

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Asp Gly Lys Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 87

<211> 413

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 87

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala Val Ile Leu

1 5 10 15

Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln Leu Met Arg

20 25 30

Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro Leu Gly Tyr

35 40 45

Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly Gly Phe Tyr

50 55 60

Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp Asn Cys Pro

65 70 75 80

Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln Arg Thr Arg

85 90 95

Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu Cys Asp Leu

100 105 110

Thr Ile His His Gln Ser Asp Ile Lys Gln Ala Asp Pro Leu Phe His

115 120 125

Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln Val Gln Gln

130 135 140

Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu Asn Gln His

145 150 155 160

Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn Phe Pro Lys

165 170 175

Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys Asp Phe Ala

180 185 190

Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly Asn Glu Val

195 200 205

Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala Glu Ile Phe

210 215 220

Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp Gly Asn Ile

225 230 235 240

His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His Asn Ala Tyr

245 250 255

Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His Asn Gly Thr

260 265 270

Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn Ala Thr Glu

275 280 285

Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu Phe Leu Ala

290 295 300

Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu Gly Met Asp

305 310 315 320

Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu

325 330 335

Leu Phe Glu Leu Trp Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val

340 345 350

Lys Met Val Tyr Gln Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu

355 360 365

Thr Leu Lys Glu Pro Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys

370 375 380

Asp Asp Gln Thr Ala Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg

385 390 395 400

Leu Val Asn Gln Val Val Glu Pro Ala Cys Gln Leu Pro

405 410

<210> 88

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 88

Asn Asp Thr Pro Ala Ser Gly Tyr Gln Val Glu Lys Val

1 5 10

<210> 89

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 89

His Ala Glu Glu Gln Asn Gly Met Lys Leu Glu Arg Val

1 5 10

<210> 90

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 90

Ser Ala Ala Glu Pro Ala Val Arg His Leu Glu Arg Val

1 5 10

<210> 91

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 91

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Asp Lys Ala

1 5 10

<210> 92

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 92

Ser Glu Ala Ala Pro Ala Gly Tyr His Leu Gln Lys Ala

1 5 10

<210> 93

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 93

Ser Glu Ala Ala Pro Ser Gly Tyr His Leu Asp Lys Ala

1 5 10

<210> 94

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 94

Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Val Phe Glu Arg Trp

1 5 10 15

Val Asp Asn Ala Gly Lys Pro Tyr Val Ser Val Asn Met Val Tyr Gln

20 25 30

Thr Leu Ala Gln Leu His Asp Gln Ala Pro Leu Thr Leu Gln His Pro

35 40 45

Ala Gly Ser Val Arg Leu Asn Ile Pro Gly Cys Ser Asp Gln Thr Pro

50 55 60

Asp Gly Tyr Cys Pro Leu Ser Thr Phe Ser Arg Leu Val Ser His Ser

65 70 75 80

Val Glu Pro Ala Cys Gln Leu Pro

85

<210> 95

<211> 89

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 95

Gln Pro Asp Asn Thr Pro Pro Gly Gly Gly Leu Val Phe Glu Leu Trp

1 5 10 15

Gln Asn Pro Asp Asn His Gln Gln Tyr Val Ala Val Lys Met Phe Tyr

20 25 30

Gln Thr Met Asp Gln Leu Arg Asn Ser Glu Lys Leu Asp Leu Lys Ser

35 40 45

His Pro Ala Gly Ile Val Pro Ile Glu Ile Glu Gly Cys Glu Asn Ile

50 55 60

Gly Thr Asp Lys Leu Cys Gln Leu Asp Thr Phe Gln Lys Arg Val Ala

65 70 75 80

Gln Val Ile Glu Pro Ala Cys His Ile

85

<210> 96

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 96

Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Val Phe Glu Arg Leu

1 5 10 15

Ala Asp Lys Ser Gly Lys Gln Tyr Val Ser Val Ser Met Val Tyr Gln

20 25 30

Thr Leu Glu Gln Leu Arg Ser Gln Thr Pro Leu Ser Leu Asn Gln Pro

35 40 45

Ala Gly Ser Val Gln Leu Lys Ile Pro Gly Cys Asn Asp Gln Thr Ala

50 55 60

Glu Gly Tyr Cys Pro Leu Ser Thr Phe Thr Arg Val Val Ser Gln Ser

65 70 75 80

Val Glu Pro Gly Cys Gln Leu Gln

85

<210> 97

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 97

Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu Phe Glu Leu Trp

1 5 10 15

Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val Lys Met Val Tyr Gln

20 25 30

Thr Leu Ala Gln Leu Arg Asn Gln Thr Pro Leu Thr Leu Lys Glu Pro

35 40 45

Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys Asp Asp Gln Thr Ala

50 55 60

Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg Leu Val Asn Gln Val

65 70 75 80

Val Glu Pro Ala Cys Gln Leu Pro

85

<210> 98

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 98

Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu Phe Glu Leu Trp

1 5 10 15

Ser Asp Lys Glu Gly Thr Gln Tyr Val Ser Val Lys Met Val Tyr Gln

20 25 30

Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu Thr Leu Lys Glu Pro

35 40 45

Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys Asp Asp Gln Thr Ala

50 55 60

Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg Leu Val Asn Gln Val

65 70 75 80

Val Glu Pro Ala Cys Gln Leu Pro

85

<210> 99

<211> 88

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 99

Gln Pro Asp Asn Thr Pro Pro Gly Gly Ala Leu Leu Phe Glu Leu Trp

1 5 10 15

Ser Asp Lys Asp Gly Thr Gln Tyr Val Ser Val Lys Met Val Tyr Gln

20 25 30

Thr Leu Ala Gln Leu Arg Asn Met Thr Pro Leu Thr Leu Lys Glu Pro

35 40 45

Ala Gly Ser Val Pro Leu Lys Ile Pro Gly Cys Asp Asp Gln Thr Ala

50 55 60

Glu Gly Tyr Cys Pro Leu Asp Thr Phe Thr Arg Leu Val Asn Gln Val

65 70 75 80

Val Glu Pro Ala Cys Gln Leu Pro

85

<210> 100

<211> 312

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 100

Val Ile Leu Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln

1 5 10 15

Thr Met Arg Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro

20 25 30

Leu Gly Tyr Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly

35 40 45

Gly Phe Tyr Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp

50 55 60

Ser Cys Pro Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln

65 70 75 80

Arg Thr Arg Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu

85 90 95

Cys Asp Leu Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro

100 105 110

Leu Phe His Pro Val Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln

115 120 125

Val Gln Gln Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu

130 135 140

Asn Gln His Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn

145 150 155 160

Phe Pro Lys Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys

165 170 175

Asp Phe Ala Asn Ala Phe Pro Ser Lys Leu Asn Ile Ser Asp Asp Gly

180 185 190

Asn Glu Val Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala

195 200 205

Glu Ile Phe Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp

210 215 220

Gly Asn Ile His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His

225 230 235 240

Asn Ala Gln Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His

245 250 255

Asn Gly Thr Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn

260 265 270

Thr Thr Glu Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu

275 280 285

Phe Leu Ala Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu

290 295 300

Gly Met Thr Trp Thr Leu Pro Gly

305 310

<210> 101

<211> 312

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 101

Val Ile Leu Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln

1 5 10 15

Leu Met Arg Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro

20 25 30

Leu Gly Tyr Ile Thr Pro Arg Gly Glu His Leu Val Lys Leu Met Gly

35 40 45

Gly Phe Tyr Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp

50 55 60

Asn Cys Pro Thr Pro Asp Asp Val Tyr Val Trp Thr Asp Val Asn Gln

65 70 75 80

Arg Thr Arg Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu

85 90 95

Cys Asp Leu Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro

100 105 110

Leu Phe His Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln

115 120 125

Val Gln Gln Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu

130 135 140

Asn Gln His Tyr Arg Ala Glu Leu Ala Leu Met Ser Asn Val Leu Asn

145 150 155 160

Phe Pro Lys Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Gln Pro Cys

165 170 175

Asp Phe Ala Gln Met Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly

180 185 190

Asn Glu Val Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala

195 200 205

Glu Ile Phe Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp

210 215 220

Gly Asn Ile His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His

225 230 235 240

Asn Ala Tyr Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His

245 250 255

Asn Gly Thr Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Pro Asn

260 265 270

Ala Thr Glu Ser Lys Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu

275 280 285

Phe Leu Ala Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu

290 295 300

Gly Met Asp Trp Thr Leu Pro Gly

305 310

<210> 102

<211> 312

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 102

Val Ile Leu Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln

1 5 10 15

Leu Met Arg Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro

20 25 30

Leu Gly Tyr Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly

35 40 45

Gly Phe Tyr Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp

50 55 60

Arg Cys Pro Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln

65 70 75 80

Arg Thr Arg Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu

85 90 95

Cys Asp Leu Thr Ile His His Gln Asn Asp Ile Lys Gln Val Asp Pro

100 105 110

Leu Phe His Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln

115 120 125

Val Gln Gln Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu

130 135 140

Asn Gln His Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn

145 150 155 160

Phe Pro Lys Ser Pro Tyr Cys Arg Gln His Ser Val Glu Gln Pro Cys

165 170 175

Asp Phe Ala Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly

180 185 190

Asn Glu Val Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala

195 200 205

Glu Ile Phe Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp

210 215 220

Gly Asn Ile His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His

225 230 235 240

Asn Ala Gln Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His

245 250 255

Gln Gly Thr Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn

260 265 270

Thr Thr Glu Ser Lys Leu Pro Asp Ile Ser Pro Ser Val Lys Ile Leu

275 280 285

Phe Leu Ala Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu

290 295 300

Gly Met Thr Trp Thr Leu Pro Gly

305 310

<210> 103

<211> 312

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 103

Val Ile Leu Ser Arg His Gly Val Arg Ala Pro Thr Lys Met Thr Gln

1 5 10 15

Leu Met Arg Asp Val Thr Pro Tyr Thr Trp Pro Glu Trp Pro Val Pro

20 25 30

Leu Gly Tyr Ile Thr Pro Arg Gly Glu His Leu Val Ser Leu Met Gly

35 40 45

Gly Phe Tyr Arg Gln Tyr Phe Gln Gln Gln Gly Leu Leu Ser Arg Asp

50 55 60

Arg Cys Pro Thr Ala Asn Asp Val Tyr Val Trp Thr Asp Val Asn Gln

65 70 75 80

Arg Thr Arg Lys Thr Gly Glu Ala Phe Leu Ala Gly Leu Ala Pro Glu

85 90 95

Cys Asp Leu Thr Ile His His Gln Ser Asp Ile Lys Gln Val Asp Pro

100 105 110

Leu Phe His Pro Leu Lys Ala Gly Ile Cys Ser Met Asp Lys Thr Gln

115 120 125

Val Gln Gln Ala Val Glu Lys Gln Ala Gly Met Pro Ile Asp Lys Leu

130 135 140

Asn Gln His Tyr Arg Pro Glu Leu Ala Leu Met Ser Asn Val Leu Asn

145 150 155 160

Phe Pro Lys Ser Pro Tyr Cys Gln Arg His Ser Gly Glu Lys Pro Cys

165 170 175

Asp Phe Ala Asn Ala Phe Pro Ser Tyr Leu Asn Ile Ser Asp Asp Gly

180 185 190

Asn Glu Val Gln Leu Glu Gly Ala Val Gly Leu Ser Ser Thr Leu Ala

195 200 205

Glu Ile Phe Leu Leu Glu Tyr Ala Gln Gly Met Pro Val Val Ala Trp

210 215 220

Gly Asn Ile His Ser Glu Gln Glu Trp Asn Asp Leu Leu Lys Leu His

225 230 235 240

Asn Ala Gln Phe Asp Leu Met Glu Arg Thr Pro Tyr Ile Ala Lys His

245 250 255

Asn Gly Thr Pro Leu Leu Gln Thr Ile Val Asn Ala Leu Asn Ser Asn

260 265 270

Thr Thr Ala Arg Glu Leu Pro Asp Ile Ser Pro Asp Val Lys Ile Leu

275 280 285

Phe Leu Ala Gly His Asp Thr Asn Ile Ala Asn Ile Gly Gly Met Leu

290 295 300

Gly Met Thr Trp Thr Leu Pro Gly

305 310

<210> 104

<211> 431

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 104

Met Arg Val Leu Leu Val Ala Leu Ala Leu Leu Ala Leu Ala Ala Ser

1 5 10 15

Ala Thr Ser Ala Ala Gln Ser Glu Pro Glu Leu Lys Leu Glu Ser Val

20 25 30

Val Ile Val Ser Arg His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln

35 40 45

Leu Met Gln Asp Val Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys

50 55 60

Leu Gly Glu Leu Thr Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly

65 70 75 80

His Tyr Trp Arg Gln Arg Leu Val Ala Asp Gly Leu Leu Pro Lys Cys

85 90 95

Gly Cys Pro Gln Ser Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu

100 105 110

Arg Thr Arg Lys Thr Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp

115 120 125

Cys Ala Ile Thr Val His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro

130 135 140

Leu Phe Asn Pro Leu Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn

145 150 155 160

Val Thr Asp Ala Ile Leu Glu Arg Ala Gly Gly Ser Ile Ala Asp Phe

165 170 175

Thr Gly His Tyr Gln Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn

180 185 190

Phe Pro Gln Ser Asn Leu Cys Leu Lys Arg Glu Lys Gln Asp Glu Ser

195 200 205

Cys Ser Leu Thr Gln Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp

210 215 220

Cys Val Ser Leu Thr Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu

225 230 235 240

Ile Phe Leu Leu Gln Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly

245 250 255

Arg Ile Thr Asp Ser His Gln Trp Asn Thr Leu Leu Ser Leu His Asn

260 265 270

Ala Gln Phe Asp Leu Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg

275 280 285

Ala Thr Pro Leu Leu Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro

290 295 300

Pro Gln Lys Gln Ala Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe

305 310 315 320

Ile Ala Gly His Asp Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu

325 330 335

Leu Asn Trp Thr Leu Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly

340 345 350

Glu Leu Val Phe Glu Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp

355 360 365

Ile Gln Val Ser Leu Val Phe Gln Thr Leu Gln Gln Met Arg Asp Lys

370 375 380

Thr Pro Leu Ser Leu Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu

385 390 395 400

Ala Gly Cys Glu Glu Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly

405 410 415

Phe Thr Gln Ile Val Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu

420 425 430

Claims (43)

1. An animal feed pellet or premix comprising: (a) an engineered phytase polypeptide or a fragment thereof comprising phytase activity, wherein the phytase polypeptide or the fragment thereof has at least 82% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and (b) Liquid or solid carrier. 2. The pellet of claim 1, wherein the carrier comprises one or more of water, glycerol, glycerol esters, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol. 3. The pellet of claim 1, wherein the carrier is one or more hydrocolloids selected from the group consisting of alginates, gelatin, cellulose derivatives, polysaccharides, molasses and brewer's grains. 4. The pellet of claim 1, wherein the carrier is one or more of molasses, raw molasses, brewer's spent grains, liquid fermentation byproducts, liquid corn steep liquor, liquid wheat distillers spent grains, liquid corn distillers spent grains, liquid barley distillers spent grains, grain distillers spent grains, liquid corn gluten meal, liquid byproducts from ethanol processing, liquid byproducts from grain processing, liquid byproducts from gluten production. 5. The pellet of claim 1, wherein the carrier is capable of being melted. 6. The pellet of claim 5, wherein the carrier is one or more carriers selected from the group consisting of animal oils or fats, vegetable oils or fats, triglycerides, free fatty acids, animal waxes, beeswax, lanolin, shell wax, insect wax, vegetable wax, carnauba wax, candelilla wax, bayberry wax, sugar cane wax, mineral wax, synthetic wax, natural and synthetic resins, and mixtures thereof. 7. The pellet of claim 6, wherein the fatty acid is one or more selected from the group consisting of medium chain fatty acids (MCFA), lauric acid, C8+C10 mixture, butyric acid, lactic acid, propionic acid, formic acid and succinic acid. 8. The pellet of claim 6, wherein the fat is an animal fat or oil and/or a vegetable fat or oil. 9. The pellet of claim 8, wherein the vegetable fat or oil is selected from the group consisting of canola oil, cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil, tung oil, castor oil, and rapeseed oil. 10. The pellet of claim 8, wherein the vegetable fat or oil is selected from the group consisting of fully hardened palm oil, fully hardened rapeseed oil, fully hardened cottonseed oil and fully hardened soybean oil. 11. A pellet as claimed in claim 9 or claim 10 wherein the vegetable fat or oil is palm oil or fully hardened palm oil. 12. The pellet of claim 1, wherein the liquid carrier is one or more of liquid whey, liquid lactose-free whey, liquid acid whey, liquid milk, liquid milk from industrial cleaning, liquid processed milk. 13. The pellet of any one of claims 2-12, wherein the carrier is one or more of lecithin, a lecithin glycerol mixture, or a lecithin fatty acid mixture. 14. The pellet of claim 1, wherein the carrier is one or more compounds selected from the group consisting of lysine, lysine sulfate, methionine, threonine, valine, tryptophan, arginine, histidine, isoleucine, leucine, and phenylalanine. 15. The pellet of claim 1, wherein the carrier is methionine. 16. Pellets according to claim 15, wherein the methionine is in the form of L-methionine, or in the form of a synthetic methionine source, such as OLM (i.e. DL-methionine) or all its salt forms, its analogs (e.g. 2-hydroxy-4-methylthiobutyric acid or all its salt forms), its derivatives (e.g. isopropyl 2-hydroxy-4-methylthiobutyrate or any other ester) or mixtures thereof. 17. The pellet of any one of claims 2-16, wherein the carrier is a hydrolysate of protein. 18. The pellet of any one of claims 2-17, wherein the carrier is a liquid carrier. 19. The pellet of any one of claims 2-17, wherein the carrier is a solid carrier. 20. The pellet of any one of claims 1-19, further comprising vitamins and/or minerals. 21. The pellet of any one of claims 1-20, wherein the engineered phytase polypeptide or fragment thereof is in the form of particles. 22. A method for producing an animal feed pellet or premix comprising combining (a) an engineered phytase polypeptide or a fragment thereof comprising phytase activity, wherein the phytase polypeptide or fragment thereof has at least 82% sequence identity to the amino acid sequence shown in SEQ ID NO: 1; and (b) a liquid or solid carrier. 23. The method of claim 22, wherein the carrier comprises one or more of water, glycerol, glycerides, ethylene glycol, 1,2-propylene glycol, or 1,3-propylene glycol. 24. The method of claim 22, wherein the carrier is one or more hydrocolloids selected from the group consisting of alginates, gelatin, cellulose derivatives, polysaccharides, molasses, and brewer's grains. 25. The method of claim 22, wherein the carrier is one or more of molasses, raw molasses, brewer's spent grains, liquid fermentation byproducts, liquid corn steep liquor, liquid wheat distillers spent grains, liquid corn distillers spent grains, liquid barley distillers spent grains, grain distillers spent grains, liquid corn gluten meal, liquid byproducts from ethanol processing, liquid byproducts from grain processing, liquid byproducts from gluten production. 26. The method of claim 22, wherein the carrier is capable of being melted. 27. The method of claim 26, wherein the carrier is one or more carriers selected from the group consisting of animal oils or fats, vegetable oils or fats, triglycerides, free fatty acids, animal waxes, beeswax, lanolin, shell wax, insect wax, vegetable wax, carnauba wax, candelilla wax, bayberry wax, sugar cane wax, mineral wax, synthetic wax, natural and synthetic resins, and mixtures thereof. 28. The method of claim 27, wherein the fatty acid is one or more selected from the group consisting of medium chain fatty acids (MCFA), lauric acid, C8+C10 mixture, butyric acid, lactic acid, propionic acid, formic acid and succinic acid. 29. The method of claim 27, wherein the fat is an animal fat or oil and/or a vegetable fat or oil. 30. The method of claim 29, wherein the vegetable fat or oil is selected from the group consisting of canola oil, cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil, tung oil, castor oil, and rapeseed oil. 31. The method of claim 29, wherein the vegetable fat or oil is selected from the group consisting of fully hardened palm oil, fully hardened rapeseed oil, fully hardened cottonseed oil and fully hardened soybean oil. 32. A method as claimed in claim 30 or claim 31 wherein the vegetable fat or oil is palm oil or fully hardened palm oil. 33. The method of claim 22, wherein the liquid carrier is one or more of liquid whey, liquid delactose whey, liquid acid whey, liquid milk, liquid milk from industrial cleaning, liquid processed milk. 34. The method of any one of claims 23-33, wherein the carrier is one or more of lecithin, a lecithin glycerol mixture, or a lecithin fatty acid mixture. 35. The method of claim 22, wherein the carrier is one or more compounds selected from the group consisting of lysine, lysine sulfate, methionine, threonine, valine, tryptophan, arginine, histidine, isoleucine, leucine, and phenylalanine. 36. The method of claim 22, wherein the carrier is methionine. 37. A method as claimed in claim 36, wherein the methionine is in the form of L-methionine, or in the form of a synthetic methionine source, such as OLM (i.e. DL-methionine) or all its salt forms, its analogs (e.g. 2-hydroxy-4-methylthiobutyric acid or all its salt forms), its derivatives (e.g. isopropyl 2-hydroxy-4-methylthiobutyrate or any other ester) or mixtures thereof. 38. The method of any one of claims 21-37, wherein the carrier is a hydrolysate of a protein. 39. The method of any one of claims 21-38, wherein the carrier is a liquid carrier. 40. The method of any one of claims 21-38, wherein the carrier is a solid carrier. 41. The method of any one of claims 21-40, further comprising combining vitamins and/or minerals. 42. The method of any one of claims 21-41, wherein the engineered phytase polypeptide or fragment thereof is in the form of particles. 43. The method of any one of claims 21-42, further comprising (c) granulating the combination of the phytase and a carrier.

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